Phase-correction of radiofrequency-multiplexed signals

ABSTRACT

Aspects of the present disclosure include methods for characterizing particles of a sample in a flow stream. Methods according to certain embodiments include generating frequency-encoded fluorescence data from a particle of a sample in a flow stream; and calculating phase-corrected spatial data of the particle by performing a transform of the frequency-encoded fluorescence data with a phase correction component. In certain embodiments, methods include generating an image of the particle in the flow stream based on the phase-corrected spatial data. Systems having a processor with memory operably coupled to the processor having instructions stored thereon, which when executed by the processor, cause the processor to calculate phase-corrected spatial data from frequency-encoded fluorescence data of a particle a flow stream are also described. Integrated circuit devices (e.g., field programmable gate arrays) having programming for practicing the subject methods are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to U.S. Provisional Patent Application Ser.No. 62/854,875 filed May 30, 2019; the disclosure of which applicationis herein incorporated by reference.

INTRODUCTION

The characterization of analytes in biological fluids has become anintegral part of medical diagnoses and assessments of overall health andwellness of a patient. Detecting analytes in biological fluids, such ashuman blood or blood derived products, can provide results that may playa role in determining a treatment protocol of a patient having a varietyof disease conditions.

Flow cytometry is a technique used to characterize and often times sortbiological material, such as cells of a blood sample or particles ofinterest in another type of biological or chemical sample. A flowcytometer typically includes a sample reservoir for receiving a fluidsample, such as a blood sample, and a sheath reservoir containing asheath fluid. The flow cytometer transports the particles (includingcells) in the fluid sample as a cell stream to a flow cell, while alsodirecting the sheath fluid to the flow cell. To characterize thecomponents of the flow stream, the flow stream is irradiated with light.Variations in the materials in the flow stream, such as morphologies orthe presence of fluorescent labels, may cause variations in the observedlight and these variations allow for characterization and separation.

To characterize the components in the flow stream, light must impinge onthe flow stream and be collected. Light sources in flow cytometers canvary from broad spectrum lamps, light emitting diodes as well as singlewavelength lasers. The light source is aligned with the flow stream andan optical response from the illuminated particles is collected andquantified.

SUMMARY

Aspects of the present disclosure include methods for characterizingparticles of a sample in a flow stream. Methods according to certainembodiments include generating frequency-encoded fluorescence data froma particle of a sample in a flow stream and calculating phase-correctedspatial data of the particle by performing a transform of thefrequency-encoded fluorescence data with a phase correction component.In certain embodiments, methods include generating an image of theparticle in the flow stream based on the phase-corrected spatial data.Systems having a processor with memory operably coupled to the processorhaving instructions stored thereon, which when executed by theprocessor, cause the processor to calculate phase-corrected spatial datafrom frequency-encoded fluorescence data of a particle a flow stream arealso described. Integrated circuit devices (e.g., field programmablegate arrays) having programming for practicing the subject methods arealso provided.

In embodiments, frequency-encoded fluorescence data from a particle in asample is generated from light detected in an interrogation region ofthe flow stream. In some embodiments, the particle is a cell. Inembodiments, methods include detecting light emission (e.g.,fluorescence) from the sample in the flow stream to generate thefrequency-encoded fluorescence data from the particle.

In some embodiments, methods further include detecting light absorption,light scatter or a combination thereof. In some embodiments, theparticle having one or more fluorophores is irradiated with a pluralityof frequency shifted beams of light from a light beam generator togenerate frequency-encoded fluorescence. In one example, a plurality ofpositions across (a horizontal axis) the flow stream are irradiated by alaser beam that includes a local oscillator beam and a plurality ofradiofrequency-shifted laser beams such that different locations acrossthe flow stream are irradiated by the local oscillator beam and one ofthe radiofrequency-shifted beams. In some instances, the localoscillator is a frequency-shifted beam of light from a laser. In thisexample, each spatial location across the particle in the flow stream ischaracterized by a different beat frequency which corresponds to thedifference between the frequency of the local oscillator beam and thefrequency of the radiofrequency-shifted beam at that location. In someembodiments, frequency-encoded data from the particle includes spatiallyencoded beat frequencies across a horizontal axis of the particle in theflow stream.

In practicing the subject methods, light from the sample in a flowstream is detected in an interrogation region and frequency encoded datafrom a particle in the sample is generated. In some embodiments, theparticles detected in the interrogation region include cells. In someembodiments, methods include detecting one or more of light absorption,light scatter, light emission (e.g., fluorescence) from the sample inthe flow stream. In some instances, phase-corrected spatial data of oneor more particles in the sample is generated from detected lightabsorption (e.g., brightfield image data). In other instances,phase-corrected spatial data of one or more particles in the sample isgenerated from detected light scatter (e.g., forward scatter image data,side scatter image data). In yet other instances, phase-correctedspatial data of one or more particles in the sample are generated fromdetected fluorescence (e.g., fluorescent marker image data). In stillother instances, phase-corrected spatial data of one or more particlesin the sample is generated from a combination of two or more of detectedlight absorption, detected light scatter and detected fluorescence.

In embodiments, the frequency-encoded fluorescence data from theparticle in the flow stream is transformed with a phase correctioncomponent to give spatial data of the particle. In embodiments, thespatial data may include the horizontal size dimensions of the particle,the vertical size dimensions of the particle, the ratio of particle sizealong two different dimensions, the ratio size of particle components(e.g., the ratio of horizontal dimension of the nucleus to horizontaldimension of the cytoplasm of a cell). In some embodiments, thefrequency-encoded fluorescence data is transformed by a Fouriertransform of the frequency-encoded fluorescence data with the phasecorrection component. In some instances, the frequency-encodedfluorescence data is transformed by a discrete Fourier transform (DFT)of the frequency-encoded fluorescence data with the phase correctioncomponent. In other instances, the phase-corrected spatial data iscalculated by performing a short time Fourier transform (STFT) of thefrequency-encoded fluorescence data with the phase correction. In stillother instances, the phase-corrected spatial data is calculated with adigital lock-in amplifier to heterodyne and de-multiplex thefrequency-encoded fluorescence data.

In some embodiments, methods include determining a phase correctioncomponent that is used to transform the frequency-encoded fluorescencedata into the phase-corrected spatial data. In some instances, the phasecorrection component includes modified transform coefficients. Incertain embodiments, the phase correction component includes a firstphase adjustment and a second phase adjustment. In some instances, thefirst phase adjustment includes an output signal from the lightdetection system. For example, the first phase adjustment may include anoutput signal from a brightfield photodetector.

In some embodiments, the first phase adjustment is calculated by:multiplying an output signal from the brightfield photodetector with apredetermined constant signal to produce a phase adjustment value; andcalculating the arctangent of the phase adjustment value to generate thefirst phase adjustment. In these embodiments, the phase adjustment valueis a sum of all bins in a discrete Fourier transform of thefrequency-encoded fluorescence data. In certain embodiments, the firstphase adjustment is an interferometric phase adjustment. In theseembodiments, the phase adjustment includes a phase shift caused by thelight source used to irradiate the sample in the flow stream. Forexample, the light source may be a light beam generator componentconfigured to generate at least a first beam of frequency shifted lightand a second beam of frequency shifted light. The light beam generatoraccording to certain instances includes a laser (e.g., a continuous wavelaser) and an acousto-optic deflector (e.g., coupled to a direct digitalsynthesizer RF comb generator). In some embodiments, the interferometricphase adjustment includes a phase shift resulting from vibrationsbetween components of the light beam generator. In some embodiments, thesecond phase adjustment is based on a fluorescence lifetime of afluorophore in the sample. In these embodiments, the second phaseadjustment may be calculated by taking the signal from all fluorescencedetectors to determine the phases present in the signal and calculatethe second phase adjustment from the fluorescence lifetime of thefluorophore.

Methods according to certain embodiments also include sorting one ormore particles in the sample. In some embodiments, the particle isidentified as being a single cell and is sorted to a first samplecomponent collection location. In other embodiments, the particle isidentified as being a cell aggregate and is sorted to a second samplecomponent collection location. In some instances, the first samplecomponent collection location includes a sample collection container andthe second sample component collection location includes a wastecollection container.

Aspects of the present disclosure also include systems forcharacterizing particles of a sample in a flow stream (e.g., cells in abiological sample). Systems according to certain embodiments include alight source configured to irradiate a sample having particles in a flowstream, a light detection system having a photodetector and a processorhaving memory operably coupled to the processor where the memoryincludes instructions stored thereon, which when executed by theprocessor, cause the processor to: generate frequency-encodedfluorescence data from a particle in the flow stream; and calculatephased-corrected spatial data of the particle by performing a transformof the frequency-encoded fluorescence data with a phase correctioncomponent.

In embodiments, systems are configured to generate frequency-encodedfluorescence data from a particle in a sample that is irradiated withthe light source. In some embodiments, the light source includes a lightbeam generator component configured to generate at least a first beam offrequency shifted light and a second beam of frequency shifted light.The light beam generator according to certain instances includes a laser(e.g., a continuous wave laser) and an acousto-optic deflector (e.g.,coupled to a direct digital synthesizer RF comb generator). The subjectsystems include a light detection system configured to detect one ormore of light absorption, light scatter, light emission (e.g.,fluorescence) from the sample in the flow stream. In some instances, thelight detection system includes a photodetector for detecting lightabsorption (e.g., a brightfield photodetector). In other instances, thelight detection system includes a photodetector for detecting lightscatter (e.g., forward scatter detector, side scatter detector). In yetother instances, the light detection system includes a photodetector fordetecting fluorescence. In still other instances, the light detectionsystem includes a combination of two or more of: a light absorptiondetector, a light scatter detector and an emitted (e.g., fluorescence)light detector.

In embodiments, the subject systems include a processor with memoryoperably coupled to the processor such that the memory includesinstructions stored thereon, which when executed by the processor, causethe processor to calculate phased-corrected spatial data of the particleby performing a transform of the frequency-encoded fluorescence datawith a phase correction component. In embodiments, the spatial data mayinclude the horizontal size dimensions of the particle, the verticalsize dimensions of the particle, the ratio of particle size along twodifferent dimensions, the ratio size of particle components (e.g., theratio of horizontal dimension of the nucleus to horizontal dimension ofthe cytoplasm of a cell). In some embodiments, to calculate thephase-corrected spatial data, systems are configured to perform aFourier transform of the frequency-encoded fluorescence data with thephase correction component to generate the phase-corrected spatial dataof the particle. In other embodiments, systems are configured to performa discrete Fourier transform (DFT) of the frequency-encoded fluorescencedata with the phase correction component to generate the phase-correctedspatial data of the particle. In yet other embodiments, systems areconfigured to perform a short time Fourier transform (STFT) of thefrequency-encoded fluorescence data with the phase correction component.In still other embodiments, systems are configured to calculate thephase-corrected spatial data with a digital lock-in amplifier toheterodyne and de-multiplex the frequency-encoded fluorescence data.

In some embodiments, systems include a processor with memory operablycoupled to the processor such that the memory includes instructionsstored thereon, which when executed by the processor, cause theprocessor to determine a phase correction component that is used totransform the frequency-encoded fluorescence data into thephase-corrected spatial data. In some instances, the phase correctioncomponent includes modified transform coefficients. In some embodiments,systems are configured to determine the phase correction component bycalculating a first phase adjustment and a second phase adjustment. Insome instances, the first phase adjustment includes an output signalfrom the light detection system. For example, the first phase adjustmentmay include an output signal from a brightfield photodetector.

In some embodiments, systems include a processor with memory operablycoupled to the processor such that the memory includes instructionsstored thereon, which when executed by the processor, cause theprocessor to calculate the first phase adjustment by: multiplying anoutput signal from the brightfield photodetector with a predeterminedconstant signal to produce a phase adjustment value; and calculating thearctangent of the phase adjustment value to generate the first phaseadjustment. In these embodiments, the phase adjustment value is a sum ofall bins in a discrete Fourier transform of the frequency-encodedfluorescence data. In certain embodiments, the first phase adjustment isan interferometric phase adjustment. In other embodiments, systemsinclude a processor with memory operably coupled to the processor suchthat the memory includes instructions stored thereon, which whenexecuted by the processor, cause the processor to calculate the secondphase adjustment based on a fluorescence lifetime of a fluorophore inthe sample. In these embodiments, the second phase adjustment may becalculated by the subject systems by taking the signal from allfluorescence detectors to determine the phases present in the signal andcalculating the second phase adjustment from the fluorescence lifetimeof the fluorophore.

Systems of interest are, in certain instances, configured for sortingparticles of a sample (e.g., a biological sample) in the flow stream. Insome embodiments, systems further include a particle sorting componenthaving a sample fluid delivery subsystem and a sheath fluid deliverysubsystem that is in fluid communication with an inlet of the particlesorting component and one or more sample collection containers forreceiving the sorted particle from the flow stream.

Aspects of the present disclosure also include integrated circuitdevices programmed to: generate frequency-encoded fluorescence data froma particle in the flow stream; calculate phase-corrected spatial data ofthe particle by performing a transform of the frequency-encodedfluorescence data with a phase correction component. In someembodiments, integrated circuit devices are programmed to sort theparticles, such as to a sample collection container or to a wastecollection container. Integrated circuit devices of interest mayinclude, in certain instances, a field programmable gate array (FPGA),an application specific integrated circuit (ASIC) or a complexprogrammable logic device (CPLD).

Integrated circuit devices according to certain embodiments areprogrammed to generate frequency-encoded fluorescence data from aparticle in the flow stream. In some embodiments, the integrated circuitdevice is programmed to generate frequency-encoded fluorescence datafrom data signals from a light absorption detector (e.g., brightfieldimage data). In other embodiments, the integrated circuit device isprogrammed to generate frequency-encoded fluorescence data from datasignals from a light scatter detector (e.g., forward scatter image data,side scatter image data). In yet other embodiments, the integratedcircuit device is programmed to generate frequency-encoded fluorescencedata from data signals from a light emission detector (e.g., fluorescentmarker image data). In still other instances, the integrated circuitdevice is programmed to generate frequency-encoded fluorescence datafrom a combination of two or more of detected light absorption, detectedlight scatter and detected fluorescence.

In embodiments, the integrated circuit device is programmed to calculatephase-corrected spatial data of the particle by performing a transformof the frequency-encoded fluorescence data with a phase correctioncomponent. In some instances, the integrated circuit device isprogrammed to perform a Fourier transform of the frequency-encodedfluorescence data with the phase correction component to generate thephase-corrected spatial data of the particle. In other instances, theintegrated circuit device is programmed to perform a discrete Fouriertransform of the frequency-encoded fluorescence data with the phasecorrection component to generate the phase-corrected spatial data of theparticle. In yet other instances, the integrated circuit device isprogrammed to perform a short time Fourier transform of thefrequency-encoded fluorescence data with the phase correction componentto generate the phase-corrected spatial data of the particle. In stillother instances, the integrated circuit device is programmed tocalculate the phase-corrected spatial data with a digital lock-inamplifier to heterodyne and de-multiplex the frequency-encodedfluorescence data.

In some embodiments, the integrated circuit device is programmed todetermine a phase correction component that is used to transform thefrequency-encoded fluorescence data into the phase-corrected spatialdata. In some instances, the phase correction component includesmodified transform coefficients. In some embodiments, the integratedcircuit device is programmed to determine the phase correction componentby calculating a first phase adjustment and a second phase adjustment.In some instances, the first phase adjustment includes an output signalfrom the light detection system. For example, the first phase adjustmentmay include an output signal from a brightfield photodetector.

In some embodiments, the integrated circuit device is programmed tocalculate the first phase adjustment by: multiplying an output signalfrom the brightfield photodetector with a predetermined constant signalto produce a phase adjustment value; and calculating the arctangent ofthe phase adjustment value to generate the first phase adjustment. Inthese embodiments, the phase adjustment value is a sum of all bins in adiscrete Fourier transform of the frequency-encoded fluorescence data.In certain embodiments, the first phase adjustment is an interferometricphase adjustment. In other embodiments, the integrated circuit device isprogrammed to calculate the second phase adjustment based on afluorescence lifetime of a fluorophore in the sample. In theseembodiments, the second phase adjustment may be calculated by thesubject integrated circuit by taking the signal from all fluorescencedetectors to determine the phases present in the signal and calculatingthe second phase adjustment from the fluorescence lifetime of thefluorophore.

BRIEF DESCRIPTION OF THE FIGURES

The invention may be best understood from the following detaileddescription when read in conjunction with the accompanying drawings.Included in the drawings are the following figures:

FIG. 1 depicts a flow chart for generating frequency-encodedfluorescence data and calculating phase-corrected spatial data from thefrequency-encoded fluorescence data according to certain embodiments.

FIG. 2 depicts a comparison of generating an image of a particle usingphase-corrected spatial data with an image where the spatial data is notphase-corrected according to embodiments.

DETAILED DESCRIPTION

Aspects of the present disclosure include methods for characterizingparticles of a sample in a flow stream. Methods according to certainembodiments include generating frequency-encoded fluorescence data froma particle of a sample in a flow stream; and calculating phase-correctedspatial data of the particle by performing a transform of thefrequency-encoded fluorescence data with a phase correction component.In certain embodiments, methods include generating an image of theparticle in the flow stream based on the phase-corrected spatial data.Systems having a processor with memory operably coupled to the processorhaving instructions stored thereon, which when executed by theprocessor, cause the processor to calculate phase-corrected spatial datafrom frequency-encoded fluorescence data of a particle a flow stream arealso described. Integrated circuit devices (e.g., field programmablegate arrays) having programming for practicing the subject methods arealso provided.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

While the apparatus and method has or will be described for the sake ofgrammatical fluidity with functional explanations, it is to be expresslyunderstood that the claims, unless expressly formulated under 35 U.S.C.§ 112, are not to be construed as necessarily limited in any way by theconstruction of “means” or “steps” limitations, but are to be accordedthe full scope of the meaning and equivalents of the definition providedby the claims under the judicial doctrine of equivalents, and in thecase where the claims are expressly formulated under 35 U.S.C. § 112 areto be accorded full statutory equivalents under 35 U.S.C. § 112.

As summarized above, the present disclosure provides systems and methodsfor characterizing (e.g., imaging) a particle of a sample in a flowstream. In further describing embodiments of the disclosure, methods forgenerating frequency-encoded fluorescence data from a particle of asample in a flow stream and calculating phase-corrected spatial data ofthe particle are first described in greater detail. Next, systems forcharacterizing the particles in the flow stream and separating particlesin a sample in real time are described. Integrated circuit devices, suchas field programmable gate arrays having programming for generatingfrequency-encoded fluorescence data from a particle of a sample in aflow stream and calculating phase-corrected spatial data of the particleare also provided.

Methods for Characterizing Particles in a Sample

Aspects of the present disclosure include methods for characterizingparticles of a sample (e.g., cells in a biological sample). Inpracticing methods according to certain embodiments, a sample havingcells in a flow stream is irradiated with a light source and light fromthe sample is detected to generate frequency-encoded fluorescence datafrom a particle and calculating phase-corrected spatial data of theparticle by performing a transform of the frequency-encoded fluorescencedata with a phase correction component. In some embodiments, the sampleis a biological sample. The term “biological sample” is used in itsconventional sense to refer to a whole organism, plant, fungi or asubset of animal tissues, cells or component parts which may in certaininstances be found in blood, mucus, lymphatic fluid, synovial fluid,cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid,amniotic cord blood, urine, vaginal fluid and semen. As such, a“biological sample” refers to both the native organism or a subset ofits tissues as well as to a homogenate, lysate or extract prepared fromthe organism or a subset of its tissues, including but not limited to,for example, plasma, serum, spinal fluid, lymph fluid, sections of theskin, respiratory, gastrointestinal, cardiovascular, and genitourinarytracts, tears, saliva, milk, blood cells, tumors, organs. Biologicalsamples may be any type of organismic tissue, including both healthy anddiseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certainembodiments, the biological sample is a liquid sample, such as blood orderivative thereof, e.g., plasma, tears, urine, semen, etc., where insome instances the sample is a blood sample, including whole blood, suchas blood obtained from venipuncture or fingerstick (where the blood mayor may not be combined with any reagents prior to assay, such aspreservatives, anticoagulants, etc.).

In certain embodiments the source of the sample is a “mammal” or“mammalian”, where these terms are used broadly to describe organismswhich are within the class mammalia, including the orders carnivore(e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), andprimates (e.g., humans, chimpanzees, and monkeys). In some instances,the subjects are humans. The methods may be applied to samples obtainedfrom human subjects of both genders and at any stage of development(i.e., neonates, infant, juvenile, adolescent, adult), where in certainembodiments the human subject is a juvenile, adolescent or adult. Whilethe present invention may be applied to samples from a human subject, itis to be understood that the methods may also be carried-out on samplesfrom other animal subjects (that is, in “non-human subjects”) such as,but not limited to, birds, mice, rats, dogs, cats, livestock and horses.

In practicing the subject methods, a sample having particles (e.g.,cells in a flow stream of a flow cytometer) is irradiated with lightfrom a light source. In some embodiments, the light source is abroadband light source, emitting light having a broad range ofwavelengths, such as for example, spanning 50 nm or more, such as 100 nmor more, such as 150 nm or more, such as 200 nm or more, such as 250 nmor more, such as 300 nm or more, such as 350 nm or more, such as 400 nmor more and including spanning 500 nm or more. For example, one suitablebroadband light source emits light having wavelengths from 200 nm to1500 nm. Another example of a suitable broadband light source includes alight source that emits light having wavelengths from 400 nm to 1000 nm.Where methods include irradiating with a broadband light source,broadband light source protocols of interest may include, but are notlimited to, a halogen lamp, deuterium arc lamp, xenon arc lamp,stabilized fiber-coupled broadband light source, a broadband LED withcontinuous spectrum, super-luminescent emitting diode, semiconductorlight emitting diode, wide spectrum LED white light source, an multi-LEDintegrated white light source, among other broadband light sources orany combination thereof.

In other embodiments, methods includes irradiating with a narrow bandlight source emitting a particular wavelength or a narrow range ofwavelengths, such as for example with a light source which emits lightin a narrow range of wavelengths like a range of 50 nm or less, such as40 nm or less, such as 30 nm or less, such as 25 nm or less, such as 20nm or less, such as 15 nm or less, such as 10 nm or less, such as 5 nmor less, such as 2 nm or less and including light sources which emit aspecific wavelength of light (i.e., monochromatic light). Where methodsinclude irradiating with a narrow band light source, narrow band lightsource protocols of interest may include, but are not limited to, anarrow wavelength LED, laser diode or a broadband light source coupledto one or more optical bandpass filters, diffraction gratings,monochromators or any combination thereof.

In certain embodiments, methods include irradiating the flow stream withone or more lasers. The type and number of lasers will vary depending onthe sample as well as desired light collected and may be a pulsed laseror continuous wave laser. For example, the laser may be a gas laser,such as a helium-neon laser, argon laser, krypton laser, xenon laser,nitrogen laser, CO₂ laser, CO laser, argon-fluorine (ArF) excimer laser,krypton-fluorine (KrF) excimer laser, xenon chlorine (XeCl) excimerlaser or xenon-fluorine (XeF) excimer laser or a combination thereof; adye laser, such as a stilbene, coumarin or rhodamine laser; ametal-vapor laser, such as a helium-cadmium (HeCd) laser, helium-mercury(HeHg) laser, helium-selenium (HeSe) laser, helium-silver (HeAg) laser,strontium laser, neon-copper (NeCu) laser, copper laser or gold laserand combinations thereof; a solid-state laser, such as a ruby laser, anNd:YAG laser, NdCrYAG laser, Er:YAG laser, Nd:YLF laser, Nd:YVO₄ laser,Nd:YCa₄O(BO₃)₃ laser, Nd:YCOB laser, titanium sapphire laser, thulim YAGlaser, ytterbium YAG laser, ytterbium₂O₃ laser or cerium doped lasersand combinations thereof; a semiconductor diode laser, optically pumpedsemiconductor laser (OPSL), or a frequency doubled- or frequency tripledimplementation of any of the above mentioned lasers.

The sample in the flow stream may be irradiated with one or more of theabove-mentioned light sources, such as 2 or more light sources, such as3 or more light sources, such as 4 or more light sources, such as 5 ormore light sources and including 10 or more light sources. The lightsource may include any combination of types of light sources. Forexample, in some embodiments, the methods include irradiating the samplein the flow stream with an array of lasers, such as an array having oneor more gas lasers, one or more dye lasers and one or more solid-statelasers.

The sample may be irradiated with wavelengths ranging from 200 nm to1500 nm, such as from 250 nm to 1250 nm, such as from 300 nm to 1000 nm,such as from 350 nm to 900 nm and including from 400 nm to 800 nm. Forexample, where the light source is a broadband light source, the samplemay be irradiated with wavelengths from 200 nm to 900 nm. In otherinstances, where the light source includes a plurality of narrow bandlight sources, the sample may be irradiated with specific wavelengths inthe range from 200 nm to 900 nm. For example, the light source may beplurality of narrow band LEDs (1 nm-25 nm) each independently emittinglight having a range of wavelengths between 200 nm to 900 nm. In otherembodiments, the narrow band light source includes one or more lasers(such as a laser array) and the sample is irradiated with specificwavelengths ranging from 200 nm to 700 nm, such as with a laser arrayhaving gas lasers, excimer lasers, dye lasers, metal vapor lasers andsolid-state laser as described above.

Where more than one light source is employed, the sample may beirradiated with the light sources simultaneously or sequentially, or acombination thereof. For example, the sample may be simultaneouslyirradiated with each of the light sources. In other embodiments, theflow stream is sequentially irradiated with each of the light sources.Where more than one light source is employed to irradiate the samplesequentially, the time each light source irradiates the sample mayindependently be 0.001 microseconds or more, such as 0.01 microsecondsor more, such as 0.1 microseconds or more, such as 1 microsecond ormore, such as 5 microseconds or more, such as 10 microseconds or more,such as 30 microseconds or more and including 60 microseconds or more.For example, methods may include irradiating the sample with the lightsource (e.g. laser) for a duration which ranges from 0.001 microsecondsto 100 microseconds, such as from 0.01 microseconds to 75 microseconds,such as from 0.1 microseconds to 50 microseconds, such as from 1microsecond to 25 microseconds and including from 5 microseconds to 10microseconds. In embodiments where sample is sequentially irradiatedwith two or more light sources, the duration sample is irradiated byeach light source may be the same or different.

The time period between irradiation by each light source may also vary,as desired, being separated independently by a delay of 0.001microseconds or more, such as 0.01 microseconds or more, such as 0.1microseconds or more, such as 1 microsecond or more, such as 5microseconds or more, such as by 10 microseconds or more, such as by 15microseconds or more, such as by 30 microseconds or more and includingby 60 microseconds or more. For example, the time period betweenirradiation by each light source may range from 0.001 microseconds to 60microseconds, such as from 0.01 microseconds to 50 microseconds, such asfrom 0.1 microseconds to 35 microseconds, such as from 1 microsecond to25 microseconds and including from 5 microseconds to 10 microseconds. Incertain embodiments, the time period between irradiation by each lightsource is 10 microseconds. In embodiments where sample is sequentiallyirradiated by more than two (i.e., 3 or more) light sources, the delaybetween irradiation by each light source may be the same or different.

The sample may be irradiated continuously or in discrete intervals. Insome instances, methods include irradiating the sample in the samplewith the light source continuously. In other instances, the sample in isirradiated with the light source in discrete intervals, such asirradiating every 0.001 millisecond, every 0.01 millisecond, every 0.1millisecond, every 1 millisecond, every 10 milliseconds, every 100milliseconds and including every 1000 milliseconds, or some otherinterval.

Depending on the light source, the sample may be irradiated from adistance which varies such as 0.01 mm or more, such as 0.05 mm or more,such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more,such as 2.5 mm or more, such as 5 mm or more, such as 10 mm or more,such as 15 mm or more, such as 25 mm or more and including 50 mm ormore. Also, the angle or irradiation may also vary, ranging from 10° to90°, such as from 15° to 85°, such as from 20° to 80°, such as from 25°to 75° and including from 30° to 60°, for example at a 90° angle.

In practicing the subject methods, light from the irradiated sample ismeasured, such as by collecting light from the sample over a range ofwavelengths (e.g., 200 nm-1000 nm). In embodiments, methods may includeone or more of measuring light absorption by the sample (e.g.,brightfield light data), measuring light scatter (e.g., forward or sidescatter light data) and measuring light emission by the sample (e.g.,fluorescence light data).

Light from the sample may be measured at one or more wavelengths of,such as at 5 or more different wavelengths, such as at 10 or moredifferent wavelengths, such as at 25 or more different wavelengths, suchas at 50 or more different wavelengths, such as at 100 or more differentwavelengths, such as at 200 or more different wavelengths, such as at300 or more different wavelengths and including measuring the collectedlight at 400 or more different wavelengths.

Light may be collected over one or more of the wavelength ranges of 200nm-1200 nm. In some instances, methods include measuring the light fromthe sample over a range of wavelengths, such as from 200 nm to 1200 nm,such as from 300 nm to 1100 nm, such as from 400 nm to 1000 nm, such asfrom 500 nm to 900 nm and including from 600 nm to 800 nm. In otherinstances, methods include measuring collected light at one or morespecific wavelengths. For example, the collected light may be measuredat one or more of 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723nm, 780 nm, 785 nm, 647 nm, 617 nm and any combinations thereof. Incertain embodiments, methods including measuring wavelengths of lightwhich correspond to the fluorescence peak wavelength of certainfluorophores.

The collected light may be measured continuously or in discreteintervals. In some instances, methods include taking measurements of thelight continuously. In other instances, the light is measured indiscrete intervals, such as measuring light every 0.001 millisecond,every 0.01 millisecond, every 0.1 millisecond, every 1 millisecond,every 10 milliseconds, every 100 milliseconds and including every 1000milliseconds, or some other interval.

Measurements of the collected light may be taken one or more timesduring the subject methods, such as 2 or more times, such as 3 or moretimes, such as 5 or more times and including 10 or more times. Incertain embodiments, light from the sample is measured 2 or more times,with the data in certain instances being averaged.

In some embodiments, methods include further adjusting the light fromthe sample before detecting the light. For example, the light from thesample source may be passed through one or more lenses, mirrors,pinholes, slits, gratings, light refractors, and any combinationthereof. In some instances, the collected light is passed through one ormore focusing lenses, such as to reduce the profile of the light. Inother instances, the emitted light from the sample is passed through oneor more collimators to reduce light beam divergence.

In certain embodiments, methods include irradiating the sample with twoor more beams of frequency shifted light. As described above, a lightbeam generator component may be employed having a laser and anacousto-optic device for frequency shifting the laser light. In theseembodiments, methods include irradiating the acousto-optic device withthe laser. Depending on the desired wavelengths of light produced in theoutput laser beam (e.g., for use in irradiating a sample in a flowstream), the laser may have a specific wavelength that varies from 200nm to 1500 nm, such as from 250 nm to 1250 nm, such as from 300 nm to1000 nm, such as from 350 nm to 900 nm and including from 400 nm to 800nm. The acousto-optic device may be irradiated with one or more lasers,such as 2 or more lasers, such as 3 or more lasers, such as 4 or morelasers, such as 5 or more lasers and including 10 or more lasers. Thelasers may include any combination of types of lasers. For example, insome embodiments, the methods include irradiating the acousto-opticdevice with an array of lasers, such as an array having one or more gaslasers, one or more dye lasers and one or more solid-state lasers.

Where more than one laser is employed, the acousto-optic device may beirradiated with the lasers simultaneously or sequentially, or acombination thereof. For example, the acousto-optic device may besimultaneously irradiated with each of the lasers. In other embodiments,the acousto-optic device is sequentially irradiated with each of thelasers. Where more than one laser is employed to irradiate theacousto-optic device sequentially, the time each laser irradiates theacousto-optic device may independently be 0.001 microseconds or more,such as 0.01 microseconds or more, such as 0.1 microseconds or more,such as 1 microsecond or more, such as 5 microseconds or more, such as10 microseconds or more, such as 30 microseconds or more and including60 microseconds or more. For example, methods may include irradiatingthe acousto-optic device with the laser for a duration which ranges from0.001 microseconds to 100 microseconds, such as from 0.01 microsecondsto 75 microseconds, such as from 0.1 microseconds to 50 microseconds,such as from 1 microsecond to 25 microseconds and including from 5microseconds to 10 microseconds. In embodiments where the acousto-opticdevice is sequentially irradiated with two or more lasers, the durationthe acousto-optic device is irradiated by each laser may be the same ordifferent.

The time period between irradiation by each laser may also vary, asdesired, being separated independently by a delay of 0.001 microsecondsor more, such as 0.01 microseconds or more, such as 0.1 microseconds ormore, such as 1 microsecond or more, such as 5 microseconds or more,such as by 10 microseconds or more, such as by 15 microseconds or more,such as by 30 microseconds or more and including by 60 microseconds ormore. For example, the time period between irradiation by each lightsource may range from 0.001 microseconds to 60 microseconds, such asfrom 0.01 microseconds to 50 microseconds, such as from 0.1 microsecondsto 35 microseconds, such as from 1 microsecond to 25 microseconds andincluding from 5 microseconds to 10 microseconds. In certainembodiments, the time period between irradiation by each laser is 10microseconds. In embodiments where the acousto-optic device issequentially irradiated by more than two (i.e., 3 or more) lasers, thedelay between irradiation by each laser may be the same or different.

The acousto-optic device may be irradiated continuously or in discreteintervals. In some instances, methods include irradiating theacousto-optic device with the laser continuously. In other instances,the acousto-optic device is irradiated with the laser in discreteintervals, such as irradiating every 0.001 millisecond, every 0.01millisecond, every 0.1 millisecond, every 1 millisecond, every 10milliseconds, every 100 milliseconds and including every 1000milliseconds, or some other interval.

Depending on the laser, the acousto-optic device may be irradiated froma distance which varies such as 0.01 mm or more, such as 0.05 mm ormore, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm ormore, such as 2.5 mm or more, such as 5 mm or more, such as 10 mm ormore, such as 15 mm or more, such as 25 mm or more and including 50 mmor more. Also, the angle or irradiation may also vary, ranging from 10°to 90°, such as from 15° to 85°, such as from 20° to 80°, such as from25° to 75° and including from 30° to 60°, for example at a 90° angle.

In embodiments, methods include applying radiofrequency drive signals tothe acousto-optic device to generate angularly deflected laser beams.Two or more radiofrequency drive signals may be applied to theacousto-optic device to generate an output laser beam with the desirednumber of angularly deflected laser beams, such as 3 or moreradiofrequency drive signals, such as 4 or more radiofrequency drivesignals, such as 5 or more radiofrequency drive signals, such as 6 ormore radiofrequency drive signals, such as 7 or more radiofrequencydrive signals, such as 8 or more radiofrequency drive signals, such as 9or more radiofrequency drive signals, such as 10 or more radiofrequencydrive signals, such as 15 or more radiofrequency drive signals, such as25 or more radiofrequency drive signals, such as 50 or moreradiofrequency drive signals and including 100 or more radiofrequencydrive signals.

The angularly deflected laser beams produced by the radiofrequency drivesignals each have an intensity based on the amplitude of the appliedradiofrequency drive signal. In some embodiments, methods includeapplying radiofrequency drive signals having amplitudes sufficient toproduce angularly deflected laser beams with a desired intensity. Insome instances, each applied radiofrequency drive signal independentlyhas an amplitude from about 0.001 V to about 500 V, such as from about0.005 V to about 400 V, such as from about 0.01 V to about 300 V, suchas from about 0.05 V to about 200 V, such as from about 0.1 V to about100 V, such as from about 0.5 V to about 75 V, such as from about 1 V to50 V, such as from about 2 V to 40 V, such as from 3 V to about 30 V andincluding from about 5 V to about 25 V. Each applied radiofrequencydrive signal has, in some embodiments, a frequency of from about 0.001MHz to about 500 MHz, such as from about 0.005 MHz to about 400 MHz,such as from about 0.01 MHz to about 300 MHz, such as from about 0.05MHz to about 200 MHz, such as from about 0.1 MHz to about 100 MHz, suchas from about 0.5 MHz to about 90 MHz, such as from about 1 MHz to about75 MHz, such as from about 2 MHz to about 70 MHz, such as from about 3MHz to about 65 MHz, such as from about 4 MHz to about 60 MHz andincluding from about 5 MHz to about 50 MHz.

In some embodiments, to generate the frequency-encoded fluorescencedata, the sample in the flow stream is irradiated with an output laserbeam from an acousto-optic device that includes angularly deflectedlaser beams each having an intensity based on the amplitude of theapplied radiofrequency drive signal. For example, the output laser beamused to irradiate the particle in the flow stream may include 2 or moreangularly deflected laser beams, such as 3 or more, such as 4 or more,such as 5 or more, such as 6 or more, such as 7 or more, such as 8 ormore, such as 9 or more, such as 10 or more and including 25 or moreangularly deflected laser beams. In embodiments, each of the angularlydeflected laser beams have different frequencies which are shifted fromfrequency of the input laser beam by a predetermined radiofrequency.

Each angularly deflected laser beam is also spatially shifted from eachother. Depending on the applied radiofrequency drive signals and desiredirradiation profile of the output laser beam, the angularly deflectedlaser beams may be separated by 0.001 μm or more, such as by 0.005 μm ormore, such as by 0.01 μm or more, such as by 0.05 μm or more, such as by0.1 μm or more, such as by 0.5 μm or more, such as by 1 μm or more, suchas by 5 μm or more, such as by 10 μm or more, such as by 100 μm or more,such as by 500 μm or more, such as by 1000 μm or more and including by5000 μm or more. In some embodiments, the angularly deflected laserbeams overlap, such as with an adjacent angularly deflected laser beamalong a horizontal axis of the output laser beam. The overlap betweenadjacent angularly deflected laser beams (such as overlap of beam spots)may be an overlap of 0.001 μm or more, such as an overlap of 0.005 μm ormore, such as an overlap of 0.01 μm or more, such as an overlap of 0.05μm or more, such as an overlap of 0.1 μm or more, such as an overlap of0.5 μm or more, such as an overlap of 1 μm or more, such as an overlapof 5 μm or more, such as an overlap of 10 μm or more and including anoverlap of 100 μm or more.

As a particle passes through a portion of the excitation beam formed bysuperposition of two beamlets, it is exposed to a superposition of theirelectric fields. The fluorescence emitted by the particle is frequencyencoded with a beat frequency that corresponds to a difference betweenthe optical frequencies of the incident beamlets. By way of example, thefrequency-encoded fluorescence emitted by a particle passing through aleft horizontal edge of an excitation beam, which is formed via asuperposition of a first beamlet and a second beamlet, would exhibit abeat frequency corresponding to the difference between the frequenciesof the second beamlet and first beamlet, i.e., a beat frequency off_(first) beamlet-f_(second beamlet). In this manner, the positions ofthe particles passing through the excitation beam can be encoded throughthe RF beat frequencies associated with the radiation emitted by thoseparticles. In some embodiments, such encoding of the positions of theparticles can be used to normalize the intensity of the detectedradiation emitted by those particles relative to the variation of thebeam intensity, e.g., across its horizontal direction.

In some embodiments, the frequency-encoded fluorescence emitted by aparticle is the beat frequency corresponding to the difference betweenthe frequency of a local oscillator beam (f_(Lo)) and the frequency of aradiofrequency shifted beamlet. For example, the frequency-encodedfluorescence data includes a beat frequency off_(Lo)-f_(RF shifted beamlet). Where irradiation of the flow streamincludes a local oscillator which spans a width (e.g., the entirehorizontal axis) of the flow stream, the frequency-encoded fluorescencedata includes beat frequencies corresponding to the difference betweenthe frequency of the local oscillator beam (f_(Lo)) and the frequency ofeach radiofrequency shifted beamlet (f₁, f₂, f₃, f₄, f₅, f₆, etc.). Inthese embodiments, the frequency-encoded fluorescence data may include aplurality of beat frequencies each corresponding to a location acrossthe horizontal axis of the flow stream.

As discussed in greater detail below, in one operational mode, aparticle in the flow stream can be illuminated concurrently with aplurality of excitation frequencies, each of which can be obtained,e.g., by shifting the central frequency of a laser beam. Morespecifically, a plurality of sample locations can be concurrentlyilluminated by a laser beam that is generated by mixing a referencelaser beam (e.g., a local oscillator) with a plurality ofradiofrequency-shifted laser beams such that each sample location isilluminated by the reference beam and one of the radiofrequency-shiftedbeams to excite a fluorophore of interest at that location, if present.In some embodiments, the reference local oscillator can be generated viaradiofrequency shifting of a beam of light (e.g., from a laser, such asa continuous wave laser). In these embodiments, each spatial location ofthe particle in the flow stream that is irradiated with the light is“tagged” with a different beat frequency corresponding to a differencebetween the frequency of the reference beam and that of one of theradiofrequency shifted beams. In these instances, the fluorescenceradiation emitted by the fluorophore will spatially encode the beatfrequencies.

In certain instances, the flow stream is irradiated with a plurality ofbeams of frequency-shifted light and a cell in the flow stream is imagedby fluorescence imaging using radiofrequency tagged emission (FIRE) togenerate a frequency-encoded image, such as those described in Diebold,et al. Nature Photonics Vol. 7(10); 806-810 (2013) as well as describedin U.S. Pat. Nos. 9,423,353; 9,784,661 and 10,006,852 and U.S. PatentPublication Nos. 2017/0133857 and 2017/0350803, the disclosures of whichare herein incorporated by reference.

In embodiments, the frequency-encoded fluorescence data is generated bydetecting the light from the particle in the flow stream. Thefluorescence data may be generated from one or more fluorescence lightdetectors (e.g., one or more detection channels), such as 2 or more,such as 3 or more, such as 4 or more, such as 5 or more, such as 6 ormore and including 8 or more fluorescence light detectors (e.g., 8 ormore detection channels). In some embodiments, the frequency-encodedfluorescence data includes data components taken (or derived) from lightfrom other detectors, such as detected light absorption or detectedlight scatter. In some instances, one or more data components of thefrequency-encoded fluorescence data from the sample is generated fromlight absorption detected from the sample, such as from a brightfieldlight detector. As described in greater detail below, the phasecorrection component may include signals from a brightfield detectorwhich is, in certain embodiments, used to generate a phase-correctedspatial data which accounts for an interferometric phase adjustment tothe spatial data calculated from frequency-encoded fluorescence data. Inother instances, one or more data components of the frequency-encodedfluorescence data from the sample is generated from light scatterdetected from the sample, such as from a side scatter detector, aforward scatter detector or a combination of a side scatter detector andforward scatter detector.

In embodiments, methods include calculating spatial data from thefrequency-encoded fluorescence data. The spatial data according toembodiments of the disclosure is phase-corrected by performing atransform of the frequency-encoded fluorescence data with a phasecorrection component. In some embodiments, the spatial data includeshorizontal size dimensions of the particle, vertical size dimensions ofthe particle, ratio of particle size along two different dimensions,ratio size of particle components (e.g., the ratio of horizontaldimension of the nucleus to horizontal dimension of the cytoplasm of acell).

In some instances, the phase correction component is used to generatemodified transform coefficients (i.e., for transforming thefrequency-encoded data into spatial data, described below). For example,the phase correction component may include 2 or more modified transformcoefficients, such as 3 or more, such as 4 or more and including 5 ormore modified transform coefficients. Where the spatial data iscalculated by performing a Fourier transform (as described below), incertain embodiments, the phase correction component includes modifiedtransform coefficient where the Fourier transform generates only realmathematical computational components (i.e., no imaginary mathematicalcomputation components are generated)

In certain embodiments, the phase correction component includes a firstphase adjustment and a second phase adjustment. Each phase adjustmentmay be a result of a different source of phase in the frequency-encodedfluorescence data. In one example, the first phase adjustment includesan output signal from the light detection system. For example, the firstphase adjustment may include an output signal from a brightfieldphotodetector. In some embodiments, the first phase adjustment iscalculated by: multiplying an output signal from the brightfieldphotodetector with a predetermined constant signal to produce a phaseadjustment value; and calculating the arctangent of the phase adjustmentvalue to generate the first phase adjustment. In these embodiments, thephase adjustment value is a sum of all bins in a discrete Fouriertransform of the frequency-encoded fluorescence data.

In certain embodiments, the first phase adjustment is an interferometricphase adjustment. In these embodiments, the phase adjustment includes aphase shift caused by the light source used to irradiate the sample inthe flow stream. For example, the light source may be a light beamgenerator component configured to generate at least a first beam offrequency shifted light and a second beam of frequency shifted light.The light beam generator according to certain instances includes a laser(e.g., a continuous wave laser) and an acousto-optic deflector (e.g.,coupled to a direct digital synthesizer RF comb generator). In someembodiments, the interferometric phase adjustment includes a phase shiftresulting from vibrations between components of the light beamgenerator.

In some embodiments, the second phase adjustment is based on afluorescence lifetime of a fluorophore in the sample. In theseembodiments, the second phase adjustment may be calculated by taking thesignal from all fluorescence detectors to determine the phases presentin the signal and calculate the second phase adjustment from thefluorescence lifetime of the fluorophore. Depending on the specific typeof fluorophore and number of fluorophores present, one or morefluorescence lifetimes may be calculated, such as 2 or more, such as 3or more, such as 4 or more and including 5 or more fluorescencelifetimes may be calculated. In some embodiments, each fluorescentlifetime is calculated at the fluorophore peak emission wavelength. Inthese embodiments, each fluorophore lifetime may be detected andcalculated using a signal from a different detector channel.

In embodiments, methods also include calculating phase-corrected spatialdata by performing a transform of the frequency-encoded fluorescencedata with the determined phase correction component above. In someembodiments, methods include calculating the spatial data fromfrequency-encoded fluorescence data from the object. In some instances,calculating the spatial data of the object includes performing atransform of the frequency-encoded fluorescence data. In one example,the spatial data is calculated by performing a Fourier transform (FT) ofthe frequency-encoded fluorescence data. In another example, the spatialdata is calculated by performing a discrete Fourier transform (DFT) ofthe frequency-encoded fluorescence data. In yet another example, thespatial data is calculated by performing a short time Fourier transform(STFT) of the frequency-encoded fluorescence data. In still anotherexample, the spatial data is calculated with a digital lock-in amplifierto heterodyne and de-multiplex the frequency-encoded fluorescence data.By taking into account the phase correction component before performinga transform of the frequency-encoded data into spatial data, the outputof the transform is less computationally complex as compared toperforming a transform of the raw frequency data into spatial data(i.e., without first accounting for phase) In some embodiments, methodsinclude performing a transform of the frequency-encoded fluorescencedata without performing any mathematical imaginary computations (i.e.,only performing computations for mathematical real computations of thetransform) to generate spatial data from the frequency-encodedfluorescence data.

In some embodiments, methods include generating an image of a particlein the flow stream from the frequency-encoded fluorescence. In someembodiments, the image of the particle may be generated from thefrequency-encoded fluorescence in combination with detected lightabsorption, detected light scatter or a combination thereof. In certaininstances, the image of the particle is generated from only thefrequency-encoded fluorescence. In other instances, the image of theobject is generated from the frequency-encoded fluorescence and lightabsorption detected from the sample, such as from a brightfield lightdetector. In yet other instances, the image of the particle is generatedfrom the frequency-encoded fluorescence with light scatter detected fromthe sample, such as from a side scatter detector, a forward scatterdetector or a combination of a side scatter detector and forward scatterdetector. In still other instances, the image of the particle isgenerated from the frequency-encoded fluorescence and a combination ofdetected light absorption, detected light scatter and detected lightemission.

One or more images of the particle may be generated from thefrequency-encoded fluorescence data. In some embodiments, a single imageof the particle is generated from the frequency-encoded fluorescencedata. In other embodiments, two or more images of the particle aregenerated from the frequency-encoded fluorescence data, such as 3 ormore, such as 4 or more, such as 5 or more and including 10 or moreimages or a combination thereof.

As summarized above, methods of the present disclosure also includesorting the particle. In embodiments, the particle may be sorted basedon the frequency-encoded fluorescence data, the calculated spatial data,generated image, one or more determined properties of the particle(e.g., size, center of mass, eccentricity) determined from thecalculated spatial data or the generated image or some combinationthereof. The term “sorting” is used herein in its conventional sense torefer to separating components (e.g., droplets containing cells,droplets containing non-cellular particles such as biologicalmacromolecules) of a sample and in some instances, delivering theseparated components to one or more sample collection containers. Forexample, methods may include sorting 2 or more components of the sample,such as 3 or more components, such as 4 or more components, such as 5 ormore components, such as 10 or more components, such as 15 or morecomponents and including sorting 25 or more components of the sample. Insome instances, a first sample component collection location includes asample collection container and the second sample component collectionlocation includes a waste collection container.

In sorting particles from the sample in the flow stream, methods includedata acquisition (e.g., fluorescence data), analysis (determiningfrequency-encoded fluorescence data, determining phase correctioncomponents, calculating a transform of the frequency-encoded data intophase-corrected spatial data) and recording, such as with a computer,where multiple data channels record data from each detector (e.g.,scatter detectors, brightfield photodetectors or fluorescencedetectors). In these embodiments, analysis includes classifying andcounting particles such that each particle is present as a set ofdigitized parameter values. The subject systems (described below) may beset to trigger on a selected parameter in order to distinguish theparticles of interest from background and noise.

A particular subpopulation of interest (e.g., single cells) may thenfurther analyzed by “gating” based on the frequency-encoded fluorescencedata collected for the entire population. To select an appropriate gate,the data is plotted so as to obtain the best separation ofsubpopulations possible. This procedure may be performed by plottingimage moment or one or more of the determined properties (e.g., size,center of mass, eccentricity). In other embodiments, methods includeplotting forward light scatter (FSC) vs. side (i.e., orthogonal) lightscatter (SSC) on a two-dimensional dot plot. In yet other embodiments,methods include plotting one or more of the determined properties (e.g.,size, center of mass, eccentricity) against one or more of forward lightscatter (FSC) and side (i.e., orthogonal) light scatter (SSC). In stillother embodiments, methods include gating the population of particlesfor forward light scatter (FSC) and side (i.e., orthogonal) lightscatter (SSC), followed by gating based on one or more of the determinedproperties (e.g., size, center of mass, eccentricity) based on the imageof the object. In still other embodiments, methods include gating thepopulation of particles based on one or more of the determinedproperties (e.g., size, center of mass, eccentricity) based on the imageof the object, followed by gating the population of particles forforward light scatter (FSC) and side (i.e., orthogonal) light scatter(SSC).

A subpopulation of objects is then selected (i.e., those single cellswithin the gate) and particles that are not within the gate areexcluded. Where desired, the gate may be selected by drawing a linearound the desired subpopulation using a cursor on a computer screen.Only those particles within the gate are then further analyzed byplotting the other parameters for these particles, such as fluorescence.Where desired, the above analysis may be configured to yield counts ofthe particles of interest in the sample.

In some embodiments, methods for sorting components of sample includesorting particles (e.g., cells in a biological sample) with particlesorting module having deflector plates, such as described in U.S. PatentPublication No. 2017/0299493, filed on Mar. 28, 2017, the disclosure ofwhich is incorporated herein by reference. In certain embodiments, cellsof the sample are sorted using a sort decision module having a pluralityof sort decision units, such as those described in U.S. ProvisionalPatent Application No. 62/803,264, filed on Feb. 8, 2019, the disclosureof which is incorporated herein by reference.

FIG. 1 depicts a flow chart for generating frequency-encodedfluorescence data and calculating phase-corrected spatial data from thefrequency-encoded fluorescence data according to certain embodiments. Atstep 101, light (light absorption, scattered light or emission) from aparticle (e.g., cell) in a flow stream are detected. At step 102,frequency-encoded fluorescence data (e.g., frequency data from eachspatial location along the horizontal axis) of the particle isgenerated. At step 103, phase correction components, such asinterferometric phase components and fluorescence lifetime phasecomponents are determined. At step 104, phase-corrected spatial data iscalculated by performing a transform of the frequency-encodedfluorescence data, such as with a discrete Fourier transform. Thespatial data can be used to generate an image at step 105. The imagemask can then be used to generate an image mask at step 106. Two or moreimages may be used calculate co-localization of one or more features ofthe cell (e.g., cellular organelles) at step 107 or co-localization maybe calculated using the image mask at step 108.

FIG. 2 depicts a comparison of generating an image of a particle usingphase-corrected spatial data with an image where the spatial data is notphase-corrected according to certain embodiments. As shown in panel A,frequency-encoded fluorescence data is transformed into spatial data,for example, by using a Fast Fourier transform (FFT) without phasecorrection. The generated image shows poor resolution with the particlebeing obscured by background noise. In panel B, the frequency encodedfluorescence data is phase corrected with a phase adjustment componentin conjunction with FFT to generate phase-corrected spatial data. Thephase-corrected spatial data provides for enhanced resolution particleimaging which is not obscured by background noise.

Systems for Characterizing Particles in a Sample

As summarized above, aspects of the present disclosure include a systemfor characterizing particles of a sample (e.g., cells in a biologicalsample). Systems according to certain embodiments include a light sourceconfigured to irradiate a sample having particles in a flow stream, alight detection system having a photodetector and a processor havingmemory operably coupled to the processor where the memory includesinstructions stored thereon, which when executed by the processor, causethe processor to: generate frequency-encoded fluorescence data from aparticle in the flow stream; and calculate phased-corrected spatial dataof the particle by performing a transform of the frequency-encodedfluorescence data with a phase correction component.

Systems of interest include a light source configured to irradiate asample in a flow stream. In embodiments, the light source may be anysuitable broadband or narrow band source of light. Depending on thecomponents in the sample (e.g., cells, beads, non-cellular particles,etc.), the light source may be configured to emit wavelengths of lightthat vary, ranging from 200 nm to 1500 nm, such as from 250 nm to 1250nm, such as from 300 nm to 1000 nm, such as from 350 nm to 900 nm andincluding from 400 nm to 800 nm. For example, the light source mayinclude a broadband light source emitting light having wavelengths from200 nm to 900 nm. In other instances, the light source includes a narrowband light source emitting a wavelength ranging from 200 nm to 900 nm.For example, the light source may be a narrow band LED (1 nm-25 nm)emitting light having a wavelength ranging between 200 nm to 900 nm.

In some embodiments, the light source is a laser. Lasers of interest mayinclude pulsed lasers or continuous wave lasers. For example, the lasermay be a gas laser, such as a helium-neon laser, argon laser, kryptonlaser, xenon laser, nitrogen laser, CO₂ laser, CO laser, argon-fluorine(ArF) excimer laser, krypton-fluorine (KrF) excimer laser, xenonchlorine (XeCl) excimer laser or xenon-fluorine (XeF) excimer laser or acombination thereof; a dye laser, such as a stilbene, coumarin orrhodamine laser; a metal-vapor laser, such as a helium-cadmium (HeCd)laser, helium-mercury (HeHg) laser, helium-selenium (HeSe) laser,helium-silver (HeAg) laser, strontium laser, neon-copper (NeCu) laser,copper laser or gold laser and combinations thereof; a solid-statelaser, such as a ruby laser, an Nd:YAG laser, NdCrYAG laser, Er:YAGlaser, Nd:YLF laser, Nd:YVO₄ laser, Nd:YCa₄O(BO₃)₃ laser, Nd:YCOB laser,titanium sapphire laser, thulim YAG laser, ytterbium YAG laser,ytterbium₂O₃ laser or cerium doped lasers and combinations thereof; asemiconductor diode laser, optically pumped semiconductor laser (OPSL),or a frequency doubled- or frequency tripled implementation of any ofthe above mentioned lasers.

In other embodiments, the light source is a non-laser light source, suchas a lamp, including but not limited to a halogen lamp, deuterium arclamp, xenon arc lamp, a light-emitting diode, such as a broadband LEDwith continuous spectrum, superluminescent emitting diode, semiconductorlight emitting diode, wide spectrum LED white light source, an multi-LEDintegrated. In some instances the non-laser light source is a stabilizedfiber-coupled broadband light source, white light source, among otherlight sources or any combination thereof.

In certain embodiments, the light source is a light beam generator thatis configured to generate two or more beams of frequency shifted light.In some instances, the light beam generator includes a laser, aradiofrequency generator configured to apply radiofrequency drivesignals to an acousto-optic device to generate two or more angularlydeflected laser beams. In these embodiments, the laser may be a pulsedlasers or continuous wave laser. For example lasers in light beamgenerators of interest may be a gas laser, such as a helium-neon laser,argon laser, krypton laser, xenon laser, nitrogen laser, CO₂ laser, COlaser, argon-fluorine (ArF) excimer laser, krypton-fluorine (KrF)excimer laser, xenon chlorine (XeCl) excimer laser or xenon-fluorine(XeF) excimer laser or a combination thereof; a dye laser, such as astilbene, coumarin or rhodamine laser; a metal-vapor laser, such as ahelium-cadmium (HeCd) laser, helium-mercury (HeHg) laser,helium-selenium (HeSe) laser, helium-silver (HeAg) laser, strontiumlaser, neon-copper (NeCu) laser, copper laser or gold laser andcombinations thereof; a solid-state laser, such as a ruby laser, anNd:YAG laser, NdCrYAG laser, Er:YAG laser, Nd:YLF laser, Nd:YVO₄ laser,Nd:YCa₄O(BO₃)₃ laser, Nd:YCOB laser, titanium sapphire laser, thulim YAGlaser, ytterbium YAG laser, ytterbium₂O₃ laser or cerium doped lasersand combinations thereof.

The acousto-optic device may be any convenient acousto-optic protocolconfigured to frequency shift laser light using applied acoustic waves.In certain embodiments, the acousto-optic device is an acousto-opticdeflector. The acousto-optic device in the subject system is configuredto generate angularly deflected laser beams from the light from thelaser and the applied radiofrequency drive signals. The radiofrequencydrive signals may be applied to the acousto-optic device with anysuitable radiofrequency drive signal source, such as a direct digitalsynthesizer (DDS), arbitrary waveform generator (AWG), or electricalpulse generator.

In embodiments, a controller is configured to apply radiofrequency drivesignals to the acousto-optic device to produce the desired number ofangularly deflected laser beams in the output laser beam, such as beingconfigured to apply 3 or more radiofrequency drive signals, such as 4 ormore radiofrequency drive signals, such as 5 or more radiofrequencydrive signals, such as 6 or more radiofrequency drive signals, such as 7or more radiofrequency drive signals, such as 8 or more radiofrequencydrive signals, such as 9 or more radiofrequency drive signals, such as10 or more radiofrequency drive signals, such as 15 or moreradiofrequency drive signals, such as 25 or more radiofrequency drivesignals, such as 50 or more radiofrequency drive signals and includingbeing configured to apply 100 or more radiofrequency drive signals.

In some instances, to produce an intensity profile of the angularlydeflected laser beams in the output laser beam, the controller isconfigured to apply radiofrequency drive signals having an amplitudethat varies such as from about 0.001 V to about 500 V, such as fromabout 0.005 V to about 400 V, such as from about 0.01 V to about 300 V,such as from about 0.05 V to about 200 V, such as from about 0.1 V toabout 100 V, such as from about 0.5 V to about 75 V, such as from about1 V to 50 V, such as from about 2 V to 40 V, such as from 3 V to about30 V and including from about 5 V to about 25 V. Each appliedradiofrequency drive signal has, in some embodiments, a frequency offrom about 0.001 MHz to about 500 MHz, such as from about 0.005 MHz toabout 400 MHz, such as from about 0.01 MHz to about 300 MHz, such asfrom about 0.05 MHz to about 200 MHz, such as from about 0.1 MHz toabout 100 MHz, such as from about 0.5 MHz to about 90 MHz, such as fromabout 1 MHz to about 75 MHz, such as from about 2 MHz to about 70 MHz,such as from about 3 MHz to about 65 MHz, such as from about 4 MHz toabout 60 MHz and including from about 5 MHz to about 50 MHz.

In certain embodiments, the controller has a processor having memoryoperably coupled to the processor such that the memory includesinstructions stored thereon, which when executed by the processor, causethe processor to produce an output laser beam with angularly deflectedlaser beams having a desired intensity profile. For example, the memorymay include instructions to produce two or more angularly deflectedlaser beams with the same intensities, such as 3 or more, such as 4 ormore, such as 5 or more, such as 10 or more, such as 25 or more, such as50 or more and including memory may include instructions to produce 100or more angularly deflected laser beams with the same intensities. Inother embodiments, the may include instructions to produce two or moreangularly deflected laser beams with different intensities, such as 3 ormore, such as 4 or more, such as 5 or more, such as 10 or more, such as25 or more, such as 50 or more and including memory may includeinstructions to produce 100 or more angularly deflected laser beams withdifferent intensities.

In certain embodiments, the controller has a processor having memoryoperably coupled to the processor such that the memory includesinstructions stored thereon, which when executed by the processor, causethe processor to produce an output laser beam having increasingintensity from the edges to the center of the output laser beam alongthe horizontal axis. In these instances, the intensity of the angularlydeflected laser beam at the center of the output beam may range from0.1% to about 99% of the intensity of the angularly deflected laserbeams at the edge of the output laser beam along the horizontal axis,such as from 0.5% to about 95%, such as from 1% to about 90%, such asfrom about 2% to about 85%, such as from about 3% to about 80%, such asfrom about 4% to about 75%, such as from about 5% to about 70%, such asfrom about 6% to about 65%, such as from about 7% to about 60%, such asfrom about 8% to about 55% and including from about 10% to about 50% ofthe intensity of the angularly deflected laser beams at the edge of theoutput laser beam along the horizontal axis. In other embodiments, thecontroller has a processor having memory operably coupled to theprocessor such that the memory includes instructions stored thereon,which when executed by the processor, cause the processor to produce anoutput laser beam having an increasing intensity from the edges to thecenter of the output laser beam along the horizontal axis. In theseinstances, the intensity of the angularly deflected laser beam at theedges of the output beam may range from 0.1% to about 99% of theintensity of the angularly deflected laser beams at the center of theoutput laser beam along the horizontal axis, such as from 0.5% to about95%, such as from 1% to about 90%, such as from about 2% to about 85%,such as from about 3% to about 80%, such as from about 4% to about 75%,such as from about 5% to about 70%, such as from about 6% to about 65%,such as from about 7% to about 60%, such as from about 8% to about 55%and including from about 10% to about 50% of the intensity of theangularly deflected laser beams at the center of the output laser beamalong the horizontal axis. In yet other embodiments, the controller hasa processor having memory operably coupled to the processor such thatthe memory includes instructions stored thereon, which when executed bythe processor, cause the processor to produce an output laser beamhaving an intensity profile with a Gaussian distribution along thehorizontal axis. In still other embodiments, the controller has aprocessor having memory operably coupled to the processor such that thememory includes instructions stored thereon, which when executed by theprocessor, cause the processor to produce an output laser beam having atop hat intensity profile along the horizontal axis.

In embodiments, light beam generators of interest may be configured toproduce angularly deflected laser beams in the output laser beam thatare spatially separated. Depending on the applied radiofrequency drivesignals and desired irradiation profile of the output laser beam, theangularly deflected laser beams may be separated by 0.001 μm or more,such as by 0.005 μm or more, such as by 0.01 μm or more, such as by 0.05μm or more, such as by 0.1 μm or more, such as by 0.5 μm or more, suchas by 1 μm or more, such as by 5 μm or more, such as by 10 μm or more,such as by 100 μm or more, such as by 500 μm or more, such as by 1000 μmor more and including by 5000 μm or more. In some embodiments, systemsare configured to produce angularly deflected laser beams in the outputlaser beam that overlap, such as with an adjacent angularly deflectedlaser beam along a horizontal axis of the output laser beam. The overlapbetween adjacent angularly deflected laser beams (such as overlap ofbeam spots) may be an overlap of 0.001 μm or more, such as an overlap of0.005 μm or more, such as an overlap of 0.01 μm or more, such as anoverlap of 0.05 μm or more, such as an overlap of 0.1 μm or more, suchas an overlap of 0.5 μm or more, such as an overlap of 1 μm or more,such as an overlap of 5 μm or more, such as an overlap of 10 μm or moreand including an overlap of 100 μm or more.

In certain instances, light beam generators configured to generate twoor more beams of frequency shifted light include laser excitationmodules as described in U.S. Pat. Nos. 9,423,353; 9,784,661 and10,006,852 and U.S. Patent Publication Nos. 2017/0133857 and2017/0350803, the disclosures of which are herein incorporated byreference.

In embodiments, systems include a light detection system having one ormore photodetectors for detecting and measuring light from the sample.Photodetectors of interest may be configured to measure light absorption(e.g., for brightfield light data), light scatter (e.g., forward or sidescatter light data), light emission (e.g., fluorescence light data) fromthe sample or a combination thereof. Photodetectors of interest mayinclude, but are not limited to optical sensors, such as active-pixelsensors (APSs), avalanche photodiode, image sensors, charge-coupleddevices (CCDs), intensified charge-coupled devices (ICCDs), lightemitting diodes, photon counters, bolometers, pyroelectric detectors,photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes,phototransistors, quantum dot photoconductors or photodiodes andcombinations thereof, among other photodetectors. In certainembodiments, light from a sample is measured with a charge-coupleddevice (CCD), semiconductor charge-coupled devices (CCD), active pixelsensors (APS), complementary metal-oxide semiconductor (CMOS) imagesensors or N-type metal-oxide semiconductor (NMOS) image sensors.

In some embodiments, light detection systems of interest include aplurality of photodetectors. In some instances, the light detectionsystem includes a plurality of solid-state detectors such asphotodiodes. In certain instances, the light detection system includes aphotodetector array, such as an array of photodiodes. In theseembodiments, the photodetector array may include 4 or morephotodetectors, such as 10 or more photodetectors, such as 25 or morephotodetectors, such as 50 or more photodetectors, such as 100 or morephotodetectors, such as 250 or more photodetectors, such as 500 or morephotodetectors, such as 750 or more photodetectors and including 1000 ormore photodetectors. For example, the detector may be a photodiode arrayhaving 4 or more photodiodes, such as 10 or more photodiodes, such as 25or more photodiodes, such as 50 or more photodiodes, such as 100 or morephotodiodes, such as 250 or more photodiodes, such as 500 or morephotodiodes, such as 750 or more photodiodes and including 1000 or morephotodiodes.

The photodetectors may be arranged in any geometric configuration asdesired, where arrangements of interest include, but are not limited toa square configuration, rectangular configuration, trapezoidalconfiguration, triangular configuration, hexagonal configuration,heptagonal configuration, octagonal configuration, nonagonalconfiguration, decagonal configuration, dodecagonal configuration,circular configuration, oval configuration as well as irregularpatterned configurations. The photodetectors in the photodetector arraymay be oriented with respect to the other (as referenced in an X-Zplane) at an angle ranging from 10° to 180°, such as from 15° to 170°,such as from 20° to 160°, such as from 25° to 150°, such as from 30° to120° and including from 45° to 90°. The photodetector array may be anysuitable shape and may be a rectilinear shape, e.g., squares,rectangles, trapezoids, triangles, hexagons, etc., curvilinear shapes,e.g., circles, ovals, as well as irregular shapes, e.g., a parabolicbottom portion coupled to a planar top portion. In certain embodiments,the photodetector array has a rectangular-shaped active surface.

Each photodetector (e.g., photodiode) in the array may have an activesurface with a width that ranges from 5 μm to 250 μm, such as from 10 μmto 225 μm, such as from 15 μm to 200 μm, such as from 20 μm to 175 μm,such as from 25 μm to 150 μm, such as from 30 μm to 125 μm and includingfrom 50 μm to 100 μm and a length that ranges from 5 μm to 250 μm, suchas from 10 μm to 225 μm, such as from 15 μm to 200 μm, such as from 20μm to 175 μm, such as from 25 μm to 150 μm, such as from 30 μm to 125 μmand including from 50 μm to 100 μm, where the surface area of eachphotodetector (e.g., photodiode) in the array ranges from 25 to μm² to10000 μm², such as from 50 to μm² to 9000 μm², such as from 75 to μm² to8000 μm², such as from 100 to μm² to 7000 μm², such as from 150 to μm²to 6000 μm² and including from 200 to μm² to 5000 μm².

The size of the photodetector array may vary depending on the amount andintensity of the light, the number of photodetectors and the desiredsensitivity and may have a length that ranges from 0.01 mm to 100 mm,such as from 0.05 mm to 90 mm, such as from 0.1 mm to 80 mm, such asfrom 0.5 mm to 70 mm, such as from 1 mm to 60 mm, such as from 2 mm to50 mm, such as from 3 mm to 40 mm, such as from 4 mm to 30 mm andincluding from 5 mm to 25 mm. The width of the photodetector array mayalso vary, ranging from 0.01 mm to 100 mm, such as from 0.05 mm to 90mm, such as from 0.1 mm to 80 mm, such as from 0.5 mm to 70 mm, such asfrom 1 mm to 60 mm, such as from 2 mm to 50 mm, such as from 3 mm to 40mm, such as from 4 mm to 30 mm and including from 5 mm to 25 mm. Assuch, the active surface of the photodetector array may range from 0.1mm² to 10000 mm², such as from 0.5 mm² to 5000 mm², such as from 1 mm²to 1000 mm², such as from 5 mm² to 500 mm², and including from 10 mm² to100 mm².

Photodetectors of interest are configured to measure collected light atone or more wavelengths, such as at 2 or more wavelengths, such as at 5or more different wavelengths, such as at 10 or more differentwavelengths, such as at 25 or more different wavelengths, such as at 50or more different wavelengths, such as at 100 or more differentwavelengths, such as at 200 or more different wavelengths, such as at300 or more different wavelengths and including measuring light emittedby a sample in the flow stream at 400 or more different wavelengths.

In some embodiments, photodetectors are configured to measure collectedlight over a range of wavelengths (e.g., 200 nm-1000 nm). In certainembodiments, photodetectors of interest are configured to collectspectra of light over a range of wavelengths. For example, systems mayinclude one or more detectors configured to collect spectra of lightover one or more of the wavelength ranges of 200 nm-1000 nm. In yetother embodiments, detectors of interest are configured to measure lightfrom the sample in the flow stream at one or more specific wavelengths.For example, systems may include one or more detectors configured tomeasure light at one or more of 450 nm, 518 nm, 519 nm, 561 nm, 578 nm,605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm,710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm and any combinationsthereof.

The light detection system is configured to measure light continuouslyor in discrete intervals. In some instances, photodetectors of interestare configured to take measurements of the collected light continuously.In other instances, the light detection system is configured to takemeasurements in discrete intervals, such as measuring light every 0.001millisecond, every 0.01 millisecond, every 0.1 millisecond, every 1millisecond, every 10 milliseconds, every 100 milliseconds and includingevery 1000 milliseconds, or some other interval.

In embodiments, systems are configured to generate frequency-encodedfluorescence data by irradiating a sample having particles in a flowstream. In some embodiments, the light source includes a light generatorcomponent that generates a plurality of angularly deflected laser beamseach having an intensity based on the amplitude of an appliedradiofrequency drive signal (e.g., from a direct digital synthesizercoupled to an acousto-optic device). For example, the subject systemsmay include light generator component that generates 2 or more angularlydeflected laser beams, such as 3 or more, such as 4 or more, such as 5or more, such as 6 or more, such as 7 or more, such as 8 or more, suchas 9 or more, such as 10 or more and including 25 or more angularlydeflected laser beams. In embodiments, each of the angularly deflectedlaser beams have different frequencies which are shifted from frequencyof the input laser beam by a predetermined radiofrequency.

The subject systems are, according to certain embodiments, configured togenerate angularly deflected laser beam that are also spatially shiftedfrom each other. Depending on the applied radiofrequency drive signalsand desired irradiation profile of the output laser beam, the subjectsystems may be configured to generate angularly deflected laser beamsthat are separated by 0.001 μm or more, such as by 0.005 μm or more,such as by 0.01 μm or more, such as by 0.05 μm or more, such as by 0.1μm or more, such as by 0.5 μm or more, such as by 1 μm or more, such asby 5 μm or more, such as by 10 μm or more, such as by 100 μm or more,such as by 500 μm or more, such as by 1000 μm or more and including by5000 μm or more. In some embodiments, the angularly deflected laserbeams overlap, such as with an adjacent angularly deflected laser beamalong a horizontal axis of the output laser beam. The overlap betweenadjacent angularly deflected laser beams (such as overlap of beam spots)may be an overlap of 0.001 μm or more, such as an overlap of 0.005 μm ormore, such as an overlap of 0.01 μm or more, such as an overlap of 0.05μm or more, such as an overlap of 0.1 μm or more, such as an overlap of0.5 μm or more, such as an overlap of 1 μm or more, such as an overlapof 5 μm or more, such as an overlap of 10 μm or more and including anoverlap of 100 μm or more.

In some embodiments, systems include a processor having memory operablycoupled to the processor where the memory includes instructions storedthereon, which when executed by the processor, cause the processor togenerate frequency-encoded fluorescence data by calculating a differencebetween the optical frequencies of the incident overlapping beamlets oflight on the flow stream. In one example, systems include a processorhaving memory operably coupled to the processor where the memoryincludes instructions stored thereon, which when executed by theprocessor, cause the processor to calculate a beat frequency at eachlocation across a horizontal axis of the flow stream. In theseembodiments, the frequency-encoded fluorescence emitted by a particle isthe beat frequency corresponding to the difference between the frequencyof a local oscillator beam (f_(Lo)) and the frequency of aradiofrequency shifted beamlet. For example, the frequency-encodedfluorescence data includes a beat frequency off_(Lo)-f_(RF shifted beamlet). Where irradiation of the flow streamincludes a local oscillator which spans a width (e.g., the entirehorizontal axis) of the flow stream, the frequency-encoded fluorescencedata includes beat frequencies corresponding to the difference betweenthe frequency of the local oscillator beam (f_(Lo)) and the frequency ofeach radiofrequency shifted beamlet (f₁, f₂, f₃, f₄, f₅, f₆, etc.). Inthese embodiments, the frequency-encoded fluorescence data may include aplurality of beat frequencies each corresponding to a location acrossthe horizontal axis of the flow stream.

In embodiments, systems are configured to generate frequency-encodedfluorescence data from detected light from the particle in the flowstream. The fluorescence data may be generated from one or morefluorescence light detectors (e.g., one or more detection channels),such as 2 or more, such as 3 or more, such as 4 or more, such as 5 ormore, such as 6 or more and including 8 or more fluorescence lightdetectors (e.g., 8 or more detection channels). In some embodiments, thefrequency-encoded fluorescence data includes data components taken (orderived) from light from other detectors, such as detected lightabsorption or detected light scatter. In some instances, systems areconfigured to generate one or more data components of thefrequency-encoded fluorescence data from light absorption detected fromthe sample, such as from a brightfield light detector. For example,systems may be configured to generate the phase correction componentfrom signals from a brightfield detector. In certain embodiments, thesystem is configured to generate a phase-corrected spatial data whichaccounts for an interferometric phase adjustment to the spatial datacalculated from frequency-encoded fluorescence data. In other instances,systems are configured to generate one or more data components of thefrequency-encoded fluorescence data from light scatter detected from thesample, such as from a side scatter detector, a forward scatter detectoror a combination of a side scatter detector and forward scatterdetector.

In embodiments, systems include a processor having memory operablycoupled to the processor where the memory includes instructions storedthereon, which when executed by the processor, cause the processor tocalculate spatial data from the frequency-encoded fluorescence data. Thespatial data according to embodiments of the disclosure isphase-corrected by the system by performing a transform of thefrequency-encoded fluorescence data with a phase correction component.In some embodiments, the spatial data includes horizontal sizedimensions of the particle, vertical size dimensions of the particle,ratio of particle size along two different dimensions, ratio size ofparticle components (e.g., the ratio of horizontal dimension of thenucleus to horizontal dimension of the cytoplasm of a cell).

In some embodiments, systems are configured to calculate modifiedtransform coefficients for transforming the frequency-encodedfluorescence data into phase-corrected spatial data. For example, thephase correction component may include 2 or more modified transformcoefficients, such as 3 or more, such as 4 or more and including 5 ormore modified transform coefficients. Where the spatial data iscalculated by performing a Fourier transform, the phase correctioncomponent includes modified transform coefficient where the Fouriertransform generates only real mathematical computational components(i.e., no imaginary mathematical computation components are generated)

In some instances, systems are configured to determine a phasecorrection component that includes a first phase adjustment and a secondphase adjustment. Each phase adjustment may be a result of a differentsource of phase in the frequency-encoded fluorescence data. In oneexample, the first phase adjustment includes an output signal from thelight detection system. For example, the first phase adjustment mayinclude an output signal from a brightfield photodetector. In someembodiments, systems include a processor having memory operably coupledto the processor where the memory includes instructions stored thereon,which when executed by the processor, cause the processor to calculate afirst phase adjustment by: multiplying an output signal from thebrightfield photodetector with a predetermined constant signal toproduce a phase adjustment value; and calculating the arctangent of thephase adjustment value to generate the first phase adjustment. In theseembodiments, the phase adjustment value is a sum of all bins in adiscrete Fourier transform of the frequency-encoded fluorescence data.

In other instances, systems are configured to calculate a second phaseadjustment that is based on fluorescence lifetime of a fluorophore inthe sample. In these instances, systems are configured to calculate thesecond phase adjustment by taking the signal from all fluorescencedetectors to determine the phases present in the signal and calculatethe second phase adjustment from the fluorescence lifetime of thefluorophore. The subject systems may be configured to calculatefluorescence lifetimes using different detector channels, such as byusing 2 or more detection channels, such as 3 or more, such as 4 or moreand including 5 or more detector channels.

In embodiments, the subject systems include a processor with memoryoperably coupled to the processor such that the memory includesinstructions stored thereon, which when executed by the processor, causethe processor to calculate phased-corrected spatial data of the particleby performing a transform of the frequency-encoded fluorescence datawith a phase correction component. In some embodiments, to calculate thephase-corrected spatial data, systems are configured to perform aFourier transform of the frequency-encoded fluorescence data with thephase correction component to generate the phase-corrected spatial dataof the particle. In other embodiments, systems are configured to performa discrete Fourier transform of the frequency-encoded fluorescence datawith the phase correction component to generate the phase-correctedspatial data of the particle. In yet other embodiments, systems areconfigured to perform a short time Fourier transform (STFT) of thefrequency-encoded fluorescence data with the phase correction component.In yet other embodiments, systems are configured to perform a discreteFourier transform (DFT) of the frequency-encoded fluorescence data withthe phase correction component. In still other embodiments, systems areconfigured to calculate the phase-corrected spatial data with a digitallock-in amplifier to heterodyne and de-multiplex the frequency-encodedfluorescence data.

In some embodiments, systems are configured to take into account thephase correction component before performing a transform of thefrequency-encoded data into spatial data so that the output of thetransform is less computationally complex as compared to performing atransform of the raw frequency data into spatial data (i.e., withoutfirst accounting for phase). In some embodiments, systems are configuredto perform a transform of the frequency-encoded fluorescence datawithout performing any mathematical imaginary computations (i.e., onlyperforming computations for mathematical real computations of thetransform) to generate spatial data from the frequency-encodedfluorescence data.

The subject systems may be configured to generate one or more images ofa particle in the flow stream from the frequency-encoded fluorescence.In some embodiments, the image of the particle may be generated from thefrequency-encoded fluorescence in combination with detected lightabsorption, detected light scatter or a combination thereof. In certaininstances, the image of the particle is generated from only thefrequency-encoded fluorescence. In other instances, the image of theobject is generated from the frequency-encoded fluorescence and lightabsorption detected from the sample, such as from a brightfield lightdetector. In yet other instances, the image of the particle is generatedfrom the frequency-encoded fluorescence with light scatter detected fromthe sample, such as from a side scatter detector, a forward scatterdetector or a combination of a side scatter detector and forward scatterdetector. In still other instances, the image of the particle isgenerated from the frequency-encoded fluorescence and a combination ofdetected light absorption, detected light scatter and detected lightemission.

Systems according to some embodiments, may include a display andoperator input device. Operator input devices may, for example, be akeyboard, mouse, or the like. The processing module includes a processorwhich has access to a memory having instructions stored thereon forperforming the steps of the subject methods. The processing module mayinclude an operating system, a graphical user interface (GUI)controller, a system memory, memory storage devices, and input-outputcontrollers, cache memory, a data backup unit, and many other devices.The processor may be a commercially available processor or it may be oneof other processors that are or will become available. The processorexecutes the operating system and the operating system interfaces withfirmware and hardware in a well-known manner, and facilitates theprocessor in coordinating and executing the functions of variouscomputer programs that may be written in a variety of programminglanguages, such as Java, Perl, C++, other high level or low-levellanguages, as well as combinations thereof, as is known in the art. Theoperating system, typically in cooperation with the processor,coordinates and executes functions of the other components of thecomputer. The operating system also provides scheduling, input-outputcontrol, file and data management, memory management, and communicationcontrol and related services, all in accordance with known techniques.The processor may be any suitable analog or digital system. In someembodiments, the processor includes analog electronics which providefeedback control, such as for example negative feedback control.

The system memory may be any of a variety of known or future memorystorage devices. Examples include any commonly available random-accessmemory (RAM), magnetic medium such as a resident hard disk or tape, anoptical medium such as a read and write compact disc, flash memorydevices, or other memory storage device. The memory storage device maybe any of a variety of known or future devices, including a compact diskdrive, a tape drive, a removable hard disk drive, or a diskette drive.Such types of memory storage devices typically read from, and/or writeto, a program storage medium (not shown) such as, respectively, acompact disk, magnetic tape, removable hard disk, or floppy diskette.Any of these program storage media, or others now in use or that maylater be developed, may be considered a computer program product. Aswill be appreciated, these program storage media typically store acomputer software program and/or data. Computer software programs, alsocalled computer control logic, typically are stored in system memoryand/or the program storage device used in conjunction with the memorystorage device.

In some embodiments, a computer program product is described comprisinga computer usable medium having control logic (computer softwareprogram, including program code) stored therein. The control logic, whenexecuted by the processor the computer, causes the processor to performfunctions described herein. In other embodiments, some functions areimplemented primarily in hardware using, for example, a hardware statemachine. Implementation of the hardware state machine so as to performthe functions described herein will be apparent to those skilled in therelevant arts.

Memory may be any suitable device in which the processor can store andretrieve data, such as magnetic, optical, or solid-state storage devices(including magnetic or optical disks or tape or RAM, or any othersuitable device, either fixed or portable). The processor may include ageneral-purpose digital microprocessor suitably programmed from acomputer readable medium carrying necessary program code. Programmingcan be provided remotely to processor through a communication channel,or previously saved in a computer program product such as memory or someother portable or fixed computer readable storage medium using any ofthose devices in connection with memory. For example, a magnetic oroptical disk may carry the programming, and can be read by a diskwriter/reader. Systems of the invention also include programming, e.g.,in the form of computer program products, algorithms for use inpracticing the methods as described above. Programming according to thepresent invention can be recorded on computer readable media, e.g., anymedium that can be read and accessed directly by a computer. Such mediainclude, but are not limited to: magnetic storage media, such as floppydiscs, hard disc storage medium, and magnetic tape; optical storagemedia such as CD-ROM; electrical storage media such as RAM and ROM;portable flash drive; and hybrids of these categories such asmagnetic/optical storage media.

The processor may also have access to a communication channel tocommunicate with a user at a remote location. By remote location ismeant the user is not directly in contact with the system and relaysinput information to an input manager from an external device, such as acomputer connected to a Wide Area Network (“WAN”), telephone network,satellite network, or any other suitable communication channel,including a mobile telephone (i.e., smartphone).

In some embodiments, systems according to the present disclosure may beconfigured to include a communication interface. In some embodiments,the communication interface includes a receiver and/or transmitter forcommunicating with a network and/or another device. The communicationinterface can be configured for wired or wireless communication,including, but not limited to, radio frequency (RF) communication (e.g.,Radio-Frequency Identification (RFID), Zigbee communication protocols,WiFi, infrared, wireless Universal Serial Bus (USB), Ultra-Wide Band(UWB), Bluetooth® communication protocols, and cellular communication,such as code division multiple access (CDMA) or Global System for Mobilecommunications (GSM).

In one embodiment, the communication interface is configured to includeone or more communication ports, e.g., physical ports or interfaces suchas a USB port, an RS-232 port, or any other suitable electricalconnection port to allow data communication between the subject systemsand other external devices such as a computer terminal (for example, ata physician's office or in hospital environment) that is configured forsimilar complementary data communication.

In one embodiment, the communication interface is configured forinfrared communication, Bluetooth® communication, or any other suitablewireless communication protocol to enable the subject systems tocommunicate with other devices such as computer terminals and/ornetworks, communication enabled mobile telephones, personal digitalassistants, or any other communication devices which the user may use inconjunction.

In one embodiment, the communication interface is configured to providea connection for data transfer utilizing Internet Protocol (IP) througha cell phone network, Short Message Service (SMS), wireless connectionto a personal computer (PC) on a Local Area Network (LAN) which isconnected to the internet, or WiFi connection to the internet at a WiFihotspot.

In one embodiment, the subject systems are configured to wirelesslycommunicate with a server device via the communication interface, e.g.,using a common standard such as 802.11 or Bluetooth® RF protocol, or anIrDA infrared protocol. The server device may be another portabledevice, such as a smart phone, Personal Digital Assistant (PDA) ornotebook computer; or a larger device such as a desktop computer,appliance, etc. In some embodiments, the server device has a display,such as a liquid crystal display (LCD), as well as an input device, suchas buttons, a keyboard, mouse or touch-screen.

In some embodiments, the communication interface is configured toautomatically or semi-automatically communicate data stored in thesubject systems, e.g., in an optional data storage unit, with a networkor server device using one or more of the communication protocols and/ormechanisms described above.

Output controllers may include controllers for any of a variety of knowndisplay devices for presenting information to a user, whether a human ora machine, whether local or remote. If one of the display devicesprovides visual information, this information typically may be logicallyand/or physically organized as an array of picture elements. A graphicaluser interface (GUI) controller may include any of a variety of known orfuture software programs for providing graphical input and outputinterfaces between the system and a user, and for processing userinputs. The functional elements of the computer may communicate witheach other via system bus. Some of these communications may beaccomplished in alternative embodiments using network or other types ofremote communications. The output manager may also provide informationgenerated by the processing module to a user at a remote location, e.g.,over the Internet, phone or satellite network, in accordance with knowntechniques. The presentation of data by the output manager may beimplemented in accordance with a variety of known techniques. As someexamples, data may include SQL, HTML or XML documents, email or otherfiles, or data in other forms. The data may include Internet URLaddresses so that a user may retrieve additional SQL, HTML, XML, orother documents or data from remote sources. The one or more platformspresent in the subject systems may be any type of known computerplatform or a type to be developed in the future, although theytypically will be of a class of computer commonly referred to asservers. However, they may also be a main-frame computer, a workstation, or other computer type. They may be connected via any known orfuture type of cabling or other communication system including wirelesssystems, either networked or otherwise. They may be co-located or theymay be physically separated. Various operating systems may be employedon any of the computer platforms, possibly depending on the type and/ormake of computer platform chosen. Appropriate operating systems includeWindows 10, Windows NT®, Windows XP, Windows 7, Windows 8, iOS, SunSolaris, Linux, OS/400, Compaq Tru64 Unix, SGI IRIX, Siemens ReliantUnix, Ubuntu, Zorin OS and others.

In certain embodiments, the subject systems include one or more opticaladjustment components for adjusting the light such as light irradiatedonto the sample (e.g., from a laser) or light collected from the sample(e.g., fluorescence). For example, the optical adjustment may be toincrease the dimensions of the light, the focus of the light or tocollimate the light. In some instances, optical adjustment is amagnification protocol so as to increase the dimensions of the light(e.g., beam spot), such as increasing the dimensions by 5% or more, suchas by 10% or more, such as by 25% or more, such as by 50% or more andincluding increasing the dimensions by 75% or more. In otherembodiments, optical adjustment includes focusing the light so as toreduce the light dimensions, such as by 5% or greater, such as by 10% orgreater, such as by 25% or greater, such as by 50% or greater andincluding reducing the dimensions of the beam spot by 75% or greater. Incertain embodiments, optical adjustment includes collimating the light.The term “collimate” is used in its conventional sense to refer to theoptically adjusting the collinearity of light propagation or reducingdivergence by the light of from a common axis of propagation. In someinstances, collimating includes narrowing the spatial cross section of alight beam (e.g., reducing the beam profile of a laser)

In some embodiments, the optical adjustment component is a focusing lenshaving a magnification ratio of from 0.1 to 0.95, such as amagnification ratio of from 0.2 to 0.9, such as a magnification ratio offrom 0.3 to 0.85, such as a magnification ratio of from 0.35 to 0.8,such as a magnification ratio of from 0.5 to 0.75 and including amagnification ratio of from 0.55 to 0.7, for example a magnificationratio of 0.6. For example, the focusing lens is, in certain instances, adouble achromatic de-magnifying lens having a magnification ratio ofabout 0.6. The focal length of the focusing lens may vary, ranging from5 mm to 20 mm, such as from 6 mm to 19 mm, such as from 7 mm to 18 mm,such as from 8 mm to 17 mm, such as from 9 mm to 16 and including afocal length ranging from 10 mm to 15 mm. In certain embodiments, thefocusing lens has a focal length of about 13 mm.

In other embodiments, the optical adjustment component is a collimator.The collimator may be any convenient collimating protocol, such as oneor more mirrors or curved lenses or a combination thereof. For example,the collimator is in certain instances a single collimating lens. Inother instances, the collimator is a collimating mirror. In yet otherinstances, the collimator includes two lenses. In still other instances,the collimator includes a mirror and a lens. Where the collimatorincludes one or more lenses, the focal length of the collimating lensmay vary, ranging from 5 mm to 40 mm, such as from 6 mm to 37.5 mm, suchas from 7 mm to 35 mm, such as from 8 mm to 32.5 mm, such as from 9 mmto 30 mm, such as from 10 mm to 27.5 mm, such as from 12.5 mm to 25 mmand including a focal length ranging from 15 mm to 20 mm.

In some embodiments, the subject systems include a flow cell nozzlehaving a nozzle orifice configured to flow a flow stream through theflow cell nozzle. The subject flow cell nozzle has an orifice whichpropagates a fluidic sample to a sample interrogation region, where insome embodiments, the flow cell nozzle includes a proximal cylindricalportion defining a longitudinal axis and a distal frustoconical portionwhich terminates in a flat surface having the nozzle orifice that istransverse to the longitudinal axis. The length of the proximalcylindrical portion (as measured along the longitudinal axis) may varyranging from 1 mm to 15 mm, such as from 1.5 mm to 12.5 mm, such as from2 mm to 10 mm, such as from 3 mm to 9 mm and including from 4 mm to 8mm. The length of the distal frustoconical portion (as measured alongthe longitudinal axis) may also vary, ranging from 1 mm to 10 mm, suchas from 2 mm to 9 mm, such as from 3 mm to 8 mm and including from 4 mmto 7 mm. The diameter of the of the flow cell nozzle chamber may vary,in some embodiments, ranging from 1 mm to 10 mm, such as from 2 mm to 9mm, such as from 3 mm to 8 mm and including from 4 mm to 7 mm.

In certain instances, the nozzle chamber does not include a cylindricalportion and the entire flow cell nozzle chamber is frustoconicallyshaped. In these embodiments, the length of the frustoconical nozzlechamber (as measured along the longitudinal axis transverse to thenozzle orifice), may range from 1 mm to 15 mm, such as from 1.5 mm to12.5 mm, such as from 2 mm to 10 mm, such as from 3 mm to 9 mm andincluding from 4 mm to 8 mm. The diameter of the proximal portion of thefrustoconical nozzle chamber may range from 1 mm to 10 mm, such as from2 mm to 9 mm, such as from 3 mm to 8 mm and including from 4 mm to 7 mm.

In embodiments, the sample flow stream emanates from an orifice at thedistal end of the flow cell nozzle. Depending on the desiredcharacteristics of the flow stream, the flow cell nozzle orifice may beany suitable shape where cross-sectional shapes of interest include, butare not limited to: rectilinear cross sectional shapes, e.g., squares,rectangles, trapezoids, triangles, hexagons, etc., curvilinearcross-sectional shapes, e.g., circles, ovals, as well as irregularshapes, e.g., a parabolic bottom portion coupled to a planar topportion. In certain embodiments, flow cell nozzle of interest has acircular orifice. The size of the nozzle orifice may vary, in someembodiments ranging from 1 μm to 20000 μm, such as from 2 μm to 17500μm, such as from 5 μm to 15000 μm, such as from 10 μm to 12500 μm, suchas from 15 μm to 10000 μm, such as from 25 μm to 7500 μm, such as from50 μm to 5000 μm, such as from 75 μm to 1000 μm, such as from 100 μm to750 μm and including from 150 μm to 500 μm. In certain embodiments, thenozzle orifice is 100 μm.

In some embodiments, the flow cell nozzle includes a sample injectionport configured to provide a sample to the flow cell nozzle. Inembodiments, the sample injection system is configured to providesuitable flow of sample to the flow cell nozzle chamber. Depending onthe desired characteristics of the flow stream, the rate of sampleconveyed to the flow cell nozzle chamber by the sample injection portmay be 1 μL/sec or more, such as 2 μL/sec or more, such as 3 μL/sec ormore, such as 5 μL/sec or more, such as 10 μL/sec or more, such as 15μL/sec or more, such as 25 μL/sec or more, such as 50 μL/sec or more,such as 100 μL/sec or more, such as 150 μL/sec or more, such as 200μL/sec or more, such as 250 μL/sec or more, such as 300 μL/sec or more,such as 350 μL/sec or more, such as 400 μL/sec or more, such as 450μL/sec or more and including 500 μL/sec or more. For example, the sampleflow rate may range from 1 μL/sec to about 500 μL/sec, such as from 2μL/sec to about 450 μL/sec, such as from 3 μL/sec to about 400 μL/sec,such as from 4 μL/sec to about 350 μL/sec, such as from 5 μL/sec toabout 300 μL/sec, such as from 6 μL/sec to about 250 μL/sec, such asfrom 7 μL/sec to about 200 μL/sec, such as from 8 μL/sec to about 150μL/sec, such as from 9 μL/sec to about 125 μL/sec and including from 10μL/sec to about 100 μL/sec.

The sample injection port may be an orifice positioned in a wall of thenozzle chamber or may be a conduit positioned at the proximal end of thenozzle chamber. Where the sample injection port is an orifice positionedin a wall of the nozzle chamber, the sample injection port orifice maybe any suitable shape where cross-sectional shapes of interest include,but are not limited to: rectilinear cross sectional shapes, e.g.,squares, rectangles, trapezoids, triangles, hexagons, etc., curvilinearcross-sectional shapes, e.g., circles, ovals, etc., as well as irregularshapes, e.g., a parabolic bottom portion coupled to a planar topportion. In certain embodiments, the sample injection port has acircular orifice. The size of the sample injection port orifice may varydepending on shape, in certain instances, having an opening ranging from0.1 mm to 5.0 mm, e.g., 0.2 to 3.0 mm, e.g., 0.5 mm to 2.5 mm, such asfrom 0.75 mm to 2.25 mm, such as from 1 mm to 2 mm and including from1.25 mm to 1.75 mm, for example 1.5 mm.

In certain instances, the sample injection port is a conduit positionedat a proximal end of the flow cell nozzle chamber. For example, thesample injection port may be a conduit positioned to have the orifice ofthe sample injection port in line with the flow cell nozzle orifice.Where the sample injection port is a conduit positioned in line with theflow cell nozzle orifice, the cross-sectional shape of the sampleinjection tube may be any suitable shape where cross-sectional shapes ofinterest include, but are not limited to: rectilinear cross sectionalshapes, e.g., squares, rectangles, trapezoids, triangles, hexagons,etc., curvilinear cross-sectional shapes, e.g., circles, ovals, as wellas irregular shapes, e.g., a parabolic bottom portion coupled to aplanar top portion. The orifice of the conduit may vary depending onshape, in certain instances, having an opening ranging from 0.1 mm to5.0 mm, e.g., 0.2 to 3.0 mm, e.g., 0.5 mm to 2.5 mm, such as from 0.75mm to 2.25 mm, such as from 1 mm to 2 mm and including from 1.25 mm to1.75 mm, for example 1.5 mm. The shape of the tip of the sampleinjection port may be the same or different from the cross-section shapeof the sample injection tube. For example, the orifice of the sampleinjection port may include a beveled tip having a bevel angle rangingfrom 1° to 10°, such as from 2° to 9°, such as from 3° to 8°, such asfrom 4° to 7° and including a bevel angle of 5°.

In some embodiments, the flow cell nozzle also includes a sheath fluidinjection port configured to provide a sheath fluid to the flow cellnozzle. In embodiments, the sheath fluid injection system is configuredto provide a flow of sheath fluid to the flow cell nozzle chamber, forexample in conjunction with the sample to produce a laminated flowstream of sheath fluid surrounding the sample flow stream. Depending onthe desired characteristics of the flow stream, the rate of sheath fluidconveyed to the flow cell nozzle chamber by the may be 25 μL/sec ormore, such as 50 μL/sec or more, such as 75 μL/sec or more, such as 100μL/sec or more, such as 250 μL/sec or more, such as 500 μL/sec or more,such as 750 μL/sec or more, such as 1000 μL/sec or more and including2500 μL/sec or more. For example, the sheath fluid flow rate may rangefrom 1 μL/sec to about 500 μL/sec, such as from 2 μL/sec to about 450μL/sec, such as from 3 μL/sec to about 400 μL/sec, such as from 4 μL/secto about 350 μL/sec, such as from 5 μL/sec to about 300 μL/sec, such asfrom 6 μL/sec to about 250 μL/sec, such as from 7 μL/sec to about 200μL/sec, such as from 8 μL/sec to about 150 μL/sec, such as from 9 μL/secto about 125 μL/sec and including from 10 μL/sec to about 100 μL/sec.

In some embodiments, the sheath fluid injection port is an orificepositioned in a wall of the nozzle chamber. The sheath fluid injectionport orifice may be any suitable shape where cross-sectional shapes ofinterest include, but are not limited to: rectilinear cross sectionalshapes, e.g., squares, rectangles, trapezoids, triangles, hexagons,etc., curvilinear cross-sectional shapes, e.g., circles, ovals, as wellas irregular shapes, e.g., a parabolic bottom portion coupled to aplanar top portion. The size of the sample injection port orifice mayvary depending on shape, in certain instances, having an opening rangingfrom 0.1 mm to 5.0 mm, e.g., 0.2 to 3.0 mm, e.g., 0.5 mm to 2.5 mm, suchas from 0.75 mm to 2.25 mm, such as from 1 mm to 2 mm and including from1.25 mm to 1.75 mm, for example 1.5 mm.

The subject systems, in certain instances, include a sampleinterrogation region in fluid communication with the flow cell nozzleorifice. In these instances, a sample flow stream emanates from anorifice at the distal end of the flow cell nozzle and particles in theflow stream may be irradiated with a light source at the sampleinterrogation region. The size of the interrogation region may varydepending on the properties of the flow nozzle, such as the size of thenozzle orifice and sample injection port size. In embodiments, theinterrogation region may have a width that is 0.01 mm or more, such as0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as1 mm or more, such as 2 mm or more, such as 3 mm or more, such as 5 mmor more and including 10 mm or more. The length of the interrogationregion may also vary, ranging in some instances along 0.01 mm or more,such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more,such as 1.5 mm or more, such as 2 mm or more, such as 3 mm or more, suchas 5 mm or more, such as 10 or more, such as 15 mm or more, such as 20mm or more, such as 25 mm or more and including 50 mm or more.

The interrogation region may be configured to facilitate irradiation ofa planar cross-section of an emanating flow stream or may be configuredto facilitate irradiation of a diffuse field (e.g., with a diffuse laseror lamp) of a predetermined length. In some embodiments, theinterrogation region includes a transparent window that facilitatesirradiation of a predetermined length of an emanating flow stream, suchas 1 mm or more, such as 2 mm or more, such as 3 mm or more, such as 4mm or more, such as 5 mm or more and including 10 mm or more. Dependingon the light source used to irradiate the emanating flow stream (asdescribed below), the interrogation region may be configured to passlight that ranges from 100 nm to 1500 nm, such as from 150 nm to 1400nm, such as from 200 nm to 1300 nm, such as from 250 nm to 1200 nm, suchas from 300 nm to 1100 nm, such as from 350 nm to 1000 nm, such as from400 nm to 900 nm and including from 500 nm to 800 nm. As such, theinterrogation region may be formed from any transparent material whichpasses the desired range of wavelength, including but not limited tooptical glass, borosilicate glass, Pyrex glass, ultraviolet quartz,infrared quartz, sapphire as well as plastic, such as polycarbonates,polyvinyl chloride (PVC), polyurethanes, polyethers, polyamides,polyimides, or copolymers of these thermoplastics, such as PETG(glycol-modified polyethylene terephthalate), among other polymericplastic materials, including polyester, where polyesters of interest mayinclude, but are not limited to poly(alkylene terephthalates) such aspoly(ethylene terephthalate) (PET), bottle-grade PET (a copolymer madebased on monoethylene glycol, terephthalic acid, and other comonomerssuch as isophthalic acid, cyclohexene dimethanol, etc.), poly(butyleneterephthalate) (PBT), and poly(hexamethylene terephthalate);poly(alkylene adipates) such as poly(ethylene adipate),poly(1,4-butylene adipate), and poly(hexamethylene adipate);poly(alkylene suberates) such as poly(ethylene suberate); poly(alkylenesebacates) such as poly(ethylene sebacate); poly(ε-caprolactone) andpoly(β-propiolactone); poly(alkylene isophthalates) such aspoly(ethylene isophthalate); poly(alkylene2,6-naphthalene-dicarboxylates) such as poly(ethylene2,6-naphthalene-dicarboxylate); poly(alkylene sulfonyl-4,4′-dibenzoates)such as poly(ethylene sulfonyl-4,4′-dibenzoate); poly(p-phenylenealkylene dicarboxylates) such as poly(p-phenylene ethylenedicarboxylates); poly(trans-1,4-cyclohexanediyl alkylene dicarboxylates)such as poly(trans-1,4-cyclohexanediyl ethylene dicarboxylate);poly(1,4-cyclohexane-dimethylene alkylene dicarboxylates) such aspoly(1,4-cyclohexane-dimethylene ethylene dicarboxylate);poly([2.2.2]-bicyclooctane-1,4-dimethylene alkylene dicarboxylates) suchas poly([2.2.2]-bicyclooctane-1,4-dimethylene ethylene dicarboxylate);lactic acid polymers and copolymers such as (S)-polylactide,(R,S)-polylactide, poly(tetramethylglycolide), andpoly(lactide-co-glycolide); and polycarbonates of bisphenol A,3,3′-dimethylbisphenol A, 3,3′,5,5′-tetrachlorobisphenol A,3,3′,5,5′-tetramethylbisphenol A; polyamides such as poly(p-phenyleneterephthalamide); polyesters, e.g., polyethylene terephthalates, e.g.,Mylar™ polyethylene terephthalate; etc. In some embodiments, the subjectsystems include a cuvette positioned in the sample interrogation region.In embodiments, the cuvette may pass light that ranges from 100 nm to1500 nm, such as from 150 nm to 1400 nm, such as from 200 nm to 1300 nm,such as from 250 nm to 1200 nm, such as from 300 nm to 1100 nm, such asfrom 350 nm to 1000 nm, such as from 400 nm to 900 nm and including from500 nm to 800 nm.

In some embodiments, the subject systems include a particle sortingcomponent for sorting particles (e.g., cells) of the sample. In certaininstances, the particle sorting component is a particle sorting modulesuch as those described in U.S. Patent Publication No. 2017/0299493,filed on Mar. 28, 2017 and U.S. Provisional Patent Application No.62/752,793 filed on Oct. 30, 2018, the disclosures of which isincorporated herein by reference. In certain embodiments, the particlesorting component include one or more droplet deflectors such as thosedescribed in U.S. Patent Publication No. 2018/0095022, filed on Jun. 14,2017, the disclosure of which is incorporated herein by reference.

In some embodiments, the subject systems are flow cytometric systems.Suitable flow cytometry systems may include, but are not limited tothose described in Ormerod (ed.), Flow Cytometry: A Practical Approach,Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow CytometryProtocols, Methods in Molecular Biology No. 91, Humana Press (1997);Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al.(2012) Ann Clin Biochem. January; 49(pt 1):17-28; Linden, et. al., SeminThrom Hemost. 2004 Oct.; 30(5):502-11; Alison, et al. J Pathol, 2010December; 222(4):335-344; and Herbig, et al. (2007) Crit Rev Ther DrugCarrier Syst. 24(3):203-255; the disclosures of which are incorporatedherein by reference. In certain instances, flow cytometry systems ofinterest include BD Biosciences FACSCanto™ II flow cytometer, BD Accuri™flow cytometer, BD Biosciences FACSCelesta™ flow cytometer, BDBiosciences FACSLyric™ flow cytometer, BD Biosciences FACSVerse™ flowcytometer, BD Biosciences FACSymphony™ flow cytometer BD BiosciencesLSRFortessa™ flow cytometer, BD Biosciences LSRFortess™ X-20 flowcytometer and BD Biosciences FACSCalibur™ cell sorter, a BD BiosciencesFACSCount™ cell sorter, BD Biosciences FACSLyric™ cell sorter and BDBiosciences Via™ cell sorter BD Biosciences Influx™ cell sorter, BDBiosciences Jazz™ cell sorter, BD Biosciences Aria™ cell sorters and BDBiosciences FACSMelody™ cell sorter, or the like.

In some embodiments, the subject particle sorting systems are flowcytometric systems, such those described in U.S. Pat. Nos. 10,006,852;9,952,076; 9,933,341; 9,784,661; 9,726,527; 9,453,789; 9,200,334;9,097,640; 9,095,494; 9,092,034; 8,975,595; 8,753,573; 8,233,146;8,140,300; 7,544,326; 7,201,875; 7,129,505; 6,821,740; 6,813,017;6,809,804; 6,372,506; 5,700,692; 5,643,796; 5,627,040; 5,620,842;5,602,039; the disclosure of which are herein incorporated by referencein their entirety.

In certain instances, the subject systems are flow cytometry systemsconfigured for characterizing and imaging particles in a flow stream byfluorescence imaging using radiofrequency tagged emission (FIRE), suchas those described in Diebold, et al. Nature Photonics Vol. 7(10);806-810 (2013) as well as described in U.S. Pat. Nos. 9,423,353;9,784,661 and 10,006,852 and U.S. Patent Publication Nos. 2017/0133857and 2017/0350803, the disclosures of which are herein incorporated byreference.

Integrated Circuit Devices

Aspects of the present disclosure also include integrated circuitdevices programmed to: generate frequency-encoded fluorescence data froma particle in the flow stream; calculate phase-corrected spatial data ofthe particle by performing a transform of the frequency-encodedfluorescence data with a phase correction component. In someembodiments, integrated circuit devices are programmed to sort theparticles, such as to a sample collection container or to a wastecollection container. Integrated circuit devices of interest mayinclude, in certain instances, a field programmable gate array (FPGA),an application specific integrated circuit (ASIC) or a complexprogrammable logic device (CPLD).

In embodiments, the integrated circuit device is programmed to generatefrequency-encoded fluorescence data. The fluorescence data may begenerated from one or more fluorescence light detectors (e.g., one ormore detection channels), such as 2 or more, such as 3 or more, such as4 or more, such as 5 or more, such as 6 or more and including 8 or morefluorescence light detectors (e.g., 8 or more detection channels). Insome embodiments, the frequency-encoded fluorescence data includes datacomponents taken (or derived) from light from other detectors, such asdetected light absorption or detected light scatter. In some instances,systems are configured to generate one or more data components of thefrequency-encoded fluorescence data from light absorption detected fromthe sample, such as from a brightfield light detector. For example,systems may be configured to generate the phase correction componentfrom signals from a brightfield detector. In certain embodiments, thesystem is configured to generate a phase-corrected spatial data whichaccounts for an interferometric phase adjustment to the spatial datacalculated from frequency-encoded fluorescence data. In other instances,systems are configured to generate one or more data components of thefrequency-encoded fluorescence data from light scatter detected from thesample, such as from a side scatter detector, a forward scatter detectoror a combination of a side scatter detector and forward scatterdetector.

In embodiments, the subject integrated circuit devices are programmed tocalculate spatial data from the frequency-encoded fluorescence data. Thespatial data according to embodiments of the disclosure isphase-corrected by performing a transform of the frequency-encodedfluorescence data with a phase correction component. In someembodiments, the spatial data includes horizontal size dimensions of theparticle, vertical size dimensions of the particle, ratio of particlesize along two different dimensions, ratio size of particle components(e.g., the ratio of horizontal dimension of the nucleus to horizontaldimension of the cytoplasm of a cell).

In some embodiments, the integrated circuit devices are programmed tocalculate modified transform coefficients for transforming thefrequency-encoded fluorescence data into phase-corrected spatial data.For example, the phase correction component may include 2 or moremodified transform coefficients, such as 3 or more, such as 4 or moreand including 5 or more modified transform coefficients. Where thespatial data is calculated by performing a Fourier transform, the phasecorrection component may include modified transform coefficient wherethe Fourier transform generates only real mathematical computationalcomponents (i.e., no imaginary mathematical computation components aregenerated)

In some instances, the integrated circuit devices are programmed todetermine a phase correction component that includes a first phaseadjustment and a second phase adjustment. Each phase adjustment may be aresult of a different source of phase in the frequency-encodedfluorescence data. In one example, the first phase adjustment includesan output signal from the light detection system. For example, theintegrated circuit devices may be programmed determine a first phaseadjustment based on an output signal from a brightfield photodetector.In some embodiments the integrated circuit devices are programmed tocalculate a first phase adjustment by: multiplying an output signal fromthe brightfield photodetector with a predetermined constant signal toproduce a phase adjustment value; and calculating the arctangent of thephase adjustment value to generate the first phase adjustment. In theseembodiments, the phase adjustment value is a sum of all bins in adiscrete Fourier transform of the frequency-encoded fluorescence data.

In other instances, the integrated circuit devices are programmed tocalculate a second phase adjustment that is based on fluorescencelifetime of a fluorophore in the sample. In these instances, theintegrated circuit devices are programmed to calculate the second phaseadjustment by taking the signal from all fluorescence detectors todetermine the phases present in the signal and calculate the secondphase adjustment from the fluorescence lifetime of the fluorophore. Thesubject integrated circuit devices may be programmed to calculatefluorescence lifetimes using different detector channels, such as byusing 2 or more detection channels, such as 3 or more, such as 4 or moreand including 5 or more detector channels.

In embodiments, the subject integrated circuit devices are programmed tocalculate phased-corrected spatial data of the particle by performing atransform of the frequency-encoded fluorescence data with a phasecorrection component. In some embodiments, to calculate thephase-corrected spatial data, systems are configured to perform aFourier transform of the frequency-encoded fluorescence data with thephase correction component to generate the phase-corrected spatial dataof the particle. In other embodiments, integrated circuit devices areprogrammed to perform a discrete Fourier transform of thefrequency-encoded fluorescence data with the phase correction componentto generate the phase-corrected spatial data of the particle. In yetother embodiments, integrated circuit devices are programmed to performa short time Fourier transform (STFT) of the frequency-encodedfluorescence data with the phase correction component. In yet otherembodiments, integrated circuit devices are programmed to perform adiscrete Fourier transform (DFT) of the frequency-encoded fluorescencedata with the phase correction component. In still other embodiments,integrated circuit devices are programmed to calculate thephase-corrected spatial data with a digital lock-in amplifier toheterodyne and de-multiplex the frequency-encoded fluorescence data.

In certain embodiments, the integrated circuit device is programmed tomake a sorting decision (as described above) based on thefrequency-encoded fluorescence data, the calculated spatial data,generated image, one or more determined properties of the particle(e.g., size, center of mass, eccentricity) determined from thecalculated spatial data or the generated image or some combinationthereof. In these embodiments, analysis includes classifying andcounting particles such that each particle is present as a set ofdigitized parameter values. The subject integrated circuit device may beprogrammed to trigger a sorting component based on a selected parameterin order to distinguish the particles of interest from background andnoise.

Kits

Aspects of the present disclosure further include kits, where kitsinclude one or more of the integrated circuit devices described herein.In some embodiments, kits may further include programming for thesubject systems, such as in the form of a computer readable medium(e.g., flash drive, USB storage, compact disk, DVD, Blu-ray disk, etc.)or instructions for downloading the programming from an internet webprotocol or cloud server. Kits may further include instructions forpracticing the subject methods. These instructions may be present in thesubject kits in a variety of forms, one or more of which may be presentin the kit. One form in which these instructions may be present is asprinted information on a suitable medium or substrate, e.g., a piece orpieces of paper on which the information is printed, in the packaging ofthe kit, in a package insert, and the like. Yet another form of theseinstructions is a computer readable medium, e.g., diskette, compact disk(CD), portable flash drive, and the like, on which the information hasbeen recorded. Yet another form of these instructions that may bepresent is a website address which may be used via the internet toaccess the information at a removed site.

Utility

The subject systems, methods and computer systems find use in a varietyof applications where it is desirable to analyze and sort particlecomponents in a sample in a fluid medium, such as a biological sample.In some embodiments, the systems and methods described herein find usein flow cytometry characterization of biological samples labelled withfluorescent tags. In other embodiments, the systems and methods find usein spectroscopy of emitted light. In addition, the subject systems andmethods find use in increasing the obtainable signal from lightcollected from a sample (e.g., in a flow stream). Embodiments of thepresent disclosure find use where it is desirable to provide a flowcytometer with improved cell sorting accuracy, enhanced particlecollection, particle charging efficiency, more accurate particlecharging and enhanced particle deflection during cell sorting.

Embodiments of the present disclosure also find use in applicationswhere cells prepared from a biological sample may be desired forresearch, laboratory testing or for use in therapy. In some embodiments,the subject methods and devices may facilitate obtaining individualcells prepared from a target fluidic or tissue biological sample. Forexample, the subject methods and systems facilitate obtaining cells fromfluidic or tissue samples to be used as a research or diagnosticspecimen for diseases such as cancer. Likewise, the subject methods andsystems may facilitate obtaining cells from fluidic or tissue samples tobe used in therapy. Methods and devices of the present disclosure allowfor separating and collecting cells from a biological sample (e.g.,organ, tissue, tissue fragment, fluid) with enhanced efficiency and lowcost as compared to traditional flow cytometry systems.

Notwithstanding the appended claims, the disclosure is also defined bythe following clauses:

1. A method comprising:

generating frequency-encoded fluorescence data from a particle of asample in a flow stream; and

calculating phase-corrected spatial data of the particle by performing atransform of the frequency-encoded fluorescence data with a phasecorrection component.

2. The method according to clause 1, wherein the spatial data iscalculated by performing a Fourier transform of the frequency-encodedfluorescence data with the phase correction component.3. The method according to clause 2, wherein the spatial data iscalculated by performing a discrete Fourier transform of thefrequency-encoded fluorescence data with the phase correction component.4. The method according to clause 2, wherein the spatial data iscalculated by performing a short time Fourier transform (STFT) of thefrequency-encoded fluorescence data with the phase correction component.5. The method according to clause 1, wherein the spatial data iscalculated with a digital lock-in amplifier to heterodyne andde-multiplex the frequency-encoded fluorescence data.6. The method according to any one of clauses 1-5, wherein the phasecorrection component comprises modified transform coefficients that areused to transform the frequency-encoded fluorescence data into thephase-corrected spatial data.7. The method according to any one of clauses 1-6, wherein generatingthe frequency-encoded fluorescence data from the particle comprisesdetecting light from the particle in the sample with a light detectionsystem.8. The method according to clause 7, wherein the light detected from theparticle comprises light absorption, light scatter, emitted light or acombination thereof.9. The method according to clause 8, wherein light absorption isdetected with a brightfield photodetector.10. The method according to any one of clauses 8-9, wherein emittedlight is detected with a fluorescence detector.11. The method according to any one of clauses 1-10, wherein the phasecorrection component comprises a first phase adjustment and a secondphase adjustment.12. The method according to clause 11, wherein the first phaseadjustment comprises an output signal from the light detection system.13. The method according to clause 12, wherein the first phaseadjustment comprises an output signal from a brightfield photodetector.14. The method according to clause 13, further comprising calculatingthe first phase adjustment by:

multiplying an output signal from the brightfield photodetector with apredetermined constant signal to produce a phase adjustment value; and

calculating the arctangent of the phase adjustment value to generate thefirst phase adjustment.

15. The method according to clause 14, wherein the phase adjustmentvalue is a sum of all bins in a discrete Fourier transform of thefrequency-encoded fluorescence data.16. The method according to any one of clauses 11-15, wherein the firstphase adjustment is an interferometric phase adjustment.17. The method according to clause 16, wherein the interferometric phaseadjustment comprises a phase shift from a light source configured toirradiate the sample in the flow stream.18. The method according to clause 17, wherein the light sourcecomprises a light beam generator component configured to generate atleast a first beam of frequency shifted light and a second beam offrequency shifted light.19. The method according to clause 18, wherein the light beam generatorcomprises an acousto-optic deflector.20. The method according to any one clauses 18-19, wherein the lightbeam generator comprises a direct digital synthesizer (DDS) RF combgenerator.21. The method according to any one of clauses 18-20, wherein the lightbeam generator component is configured to generate a frequency-shiftedlocal oscillator beam.22. The method according to any one of clauses 17-21, wherein the lightsource comprises a laser.23. The method according to clause 22, wherein the laser is a continuouswave laser.24. The method according to any one of clauses 17-23, wherein theinterferometric phase adjustment comprises a phase shift resulting fromvibrations between components of the light source.25. The method according to any one of clauses 11-24, further comprisingcalculating the second phase adjustment based on a fluorescence lifetimeof a fluorophore in the sample.26. The method according to any one of clauses 1-25, wherein thephase-corrected spatial data of the particle is calculated from thefrequency-encoded fluorescence data by an integrated circuit device.27. The method according to clause 26, wherein the integrated circuitdevice is a field programmable gate array (FPGA).28. The method according to clause 26, wherein the integrated circuitdevice is an application specific integrated circuit (ASIC).29. The method according to clause 26, wherein the integrated circuitdevice is a complex programmable logic device (CPLD).30. The method according to any one of clauses 1-29, further comprisingirradiating the flow stream with a light source.31. The method according to clause 30, wherein the flow stream isirradiated with a light source at a wavelength from 200 nm to 800 nm.32. The method according to any one of clauses 30-31, wherein the methodcomprises irradiating the flow stream with a first beam of frequencyshifted light and second beam of frequency shifted light.33. The method according to clause 32, wherein the first beam offrequency shifted light comprises a local oscillator (LO) beam and thesecond beam of frequency shifted light comprises a radiofrequency combbeam.34. The method according to any one of clauses 32-33, furthercomprising:

applying a radiofrequency drive signal to an acousto-optic device; and

irradiating the acousto-optic device with a laser to generate the firstbeam of frequency shifted light and the second beam of frequency shiftedlight.

35. The method according to clause 34, wherein the laser is a continuouswave laser.36. The method according to any one of clauses 1-35, further comprisinggenerating an image of the particle from the phase-corrected spatialdata.37. The method according to clause 36, further comprising generating animage mask of the particle.38. The method according to any one of clauses 1-37, further comprisingsorting the particle.39. A system comprising:

a light source configured to irradiate a sample comprising particles ina flow stream;

a light detection system; and

a processor comprising memory operably coupled to the processor whereinthe memory comprises instructions stored thereon, which when executed bythe processor, cause the processor to:

-   -   generate frequency-encoded fluorescence data from a particle in        the flow stream;    -   calculate phased-corrected spatial data of the particle by        performing a transform of the frequency-encoded fluorescence        data with a phase correction component.        40. The system according to clause 39, wherein the memory        comprises instructions stored thereon, which when executed by        the processor, cause the processor to perform a Fourier        transform of the frequency-encoded fluorescence data with the        phase correction component to generate the phase-corrected        spatial data of the particle.        41. The system according to clause 40, wherein the memory        comprises instructions stored thereon, which when executed by        the processor, cause the processor to perform a discrete Fourier        transform of the frequency-encoded fluorescence data with the        phase correction component to generate the phase-corrected        spatial data of the particle.        42. The system according to clause 40, wherein the memory        comprises instructions stored thereon, which when executed by        the processor, cause the processor to perform a short time        Fourier transform (STFT) of the frequency-encoded fluorescence        data with the phase correction component.        43. The system according to clause 40, wherein the memory        comprises instructions stored thereon, which when executed by        the processor, cause the processor to calculate the        phase-corrected spatial data with a digital lock-in amplifier to        heterodyne and de-multiplex the frequency-encoded fluorescence        data.        44. The system according to any one of clauses 39-43, wherein        the memory comprises instructions stored thereon, which when        executed by the processor, cause the processor to transform the        frequency-encoded fluorescence data into the spatial data with a        phase correction component that comprises modified transform        coefficients.        45. The system according to any one of clauses 39-44, wherein        the light detection system comprises a photodetector configured        to detect one or more of light absorption, light scatter and        fluorescence.        46. The system according to clause 45, wherein the light        detection system comprises a brightfield photodetector.        47. The system according to any one of clauses 39-46, wherein        the light detection system comprises a fluorescence detector.        48. The system according to any one of clauses 44-47, wherein        the memory comprises instructions stored thereon, which when        executed by the processor, cause the processor to calculate a        phase correction component that comprises a first phase        adjustment and a second phase adjustment.        49. The system according to clause 48, wherein the memory        comprises instructions stored thereon, which when executed by        the processor, cause the processor to calculate the first phase        adjustment by:

multiplying an output signal from the brightfield photo photodetectorwith a predetermined constant signal to produce a phase adjustmentvalue; and

calculating the arctangent of the phase adjustment value to generate thefirst phase adjustment.

50. The system according to clause 49, wherein the phase adjustmentvalue is a sum of all bins in a discrete Fourier transform of thefrequency-encoded fluorescence data.51. The system according to any one of clauses 48-50, wherein the firstphase adjustment is an interferometric phase adjustment.52. The system according to clause 51, wherein the interferometric phaseadjustment comprises a phase shift from the light source.53. The system according to any one of clauses 39-52, wherein the lightsource comprises a light beam generator component configured to generateat least a first beam of frequency shifted light and a second beam offrequency shifted light.54. The system according to clause 53, wherein the light beam generatorcomprises an acousto-optic deflector.55. The system according to any one clauses 53-54, wherein the lightbeam generator comprises a direct digital synthesizer (DDS) RF combgenerator.56. The system according to any one of clauses 53-55, wherein the lightbeam generator component is configured to generate a frequency-shiftedlocal oscillator beam.57. The system according to any one of clauses 39-56, wherein the lightsource comprises a laser.58. The system according to clause 57, wherein the laser is a continuouswave laser.59. The system according to any one of clauses 51-58, wherein theinterferometric phase adjustment comprises a phase shift resulting fromvibrations between components of the light source.60. The system according to any one of clauses 39-59, wherein the memorycomprises instructions stored thereon, which when executed by theprocessor, cause the processor to calculate the second phase adjustmentbased on a fluorescence lifetime of a fluorophore in the sample.61. The system according to any one of clauses 39-60, comprising anintegrated circuit component programmed for:

generating the frequency-encoded fluorescence data from the particle ofa sample in the flow stream;

calculating phase-corrected spatial data of the particle by performing atransform of the frequency-encoded fluorescence data with a phasecorrection component.

62. The system according to clause 61, wherein the integrated circuitdevice is a field programmable gate array (FPGA).63. The system according to clause 61, wherein the integrated circuitdevice is an application specific integrated circuit (ASIC).64. The system according to clause 61, wherein the integrated circuitdevice is a complex programmable logic device (CPLD).65. The system according to any one of clauses 39-64, wherein the systemis a flow cytometer.66. The system according to any one of clauses 39-65, wherein the memorycomprises instructions stored thereon, which when executed by theprocessor, cause the processor to generate an image of the particle fromthe phase-corrected spatial data.67. The system according to clause 66, wherein the memory comprisesinstructions stored thereon, which when executed by the processor, causethe processor to generate an image mask of the particle.68. The system according to any one of clauses 39-67, further comprisinga cell sorting component configured to sort cells in the sample based onthe calculated phase-corrected spatial data.69. The system according to clause 68, wherein the cell sortingcomponent comprises a droplet deflector.70. An integrated circuit programmed to:

generate frequency-encoded fluorescence data from a particle in the flowstream;

calculate phase-corrected spatial data of the particle by performing atransform of the frequency-encoded fluorescence data with a phasecorrection component.

71. The integrated circuit according to clause 70, wherein theintegrated circuit is programmed to perform a Fourier transform of thefrequency-encoded fluorescence data with the phase correction componentto generate the phase-corrected spatial data of the particle.72. The integrated circuit according to clause 71, wherein theintegrated circuit is programmed to perform a discrete Fourier transformof the frequency-encoded fluorescence data with the phase correctioncomponent to generate the phase-corrected spatial data of the particle.73. The integrated circuit according to clause 71, wherein theintegrated circuit is programmed to perform a short time Fouriertransform of the frequency-encoded fluorescence data with the phasecorrection component to generate the phase-corrected spatial data of theparticle.74. The integrated circuit according to clause 70, wherein theintegrated circuit is programmed to calculate the phase-correctedspatial data with a digital lock-in amplifier to heterodyne andde-multiplex the frequency-encoded fluorescence data.75. The integrated circuit according any one of clauses 70-74, whereinthe integrated circuit is programmed to transform the frequency-encodedfluorescence data into the spatial data with a phase correctioncomponent that comprises modified transform coefficients.76. The integrated circuit according any one of clauses 70-75, whereinthe integrated circuit is programmed to calculate a phase correctioncomponent that comprises a first phase adjustment and a second phaseadjustment.77. The integrated circuit according clause 76, wherein the integratedcircuit is programmed to calculate the first phase adjustment by:

multiplying an output signal from the brightfield photo photodetectorwith a predetermined constant signal to produce a phase adjustmentvalue; and

calculating the arctangent of the phase adjustment value to generate thefirst phase adjustment.

78. The integrated circuit according to clause 77, wherein the phaseadjustment value is a sum of all bins in a discrete Fourier transform ofthe frequency-encoded fluorescence data.79. The integrated circuit according to any one of clauses 76-78,wherein the first phase adjustment is an interferometric phaseadjustment.80. The integrated circuit according to clause 79, wherein theinterferometric phase adjustment comprises a phase shift from a lightsource configured to irradiate the sample in the flow stream.81. The integrated circuit according to clause 80, wherein the lightsource comprises a light beam generator component configured to generateat least a first beam of frequency shifted light and a second beam offrequency shifted light.82. The integrated circuit according to clause 81, wherein the lightbeam generator comprises an acousto-optic deflector.83. The integrated circuit according to any one clauses 81-82, whereinthe light beam generator comprises a direct digital synthesizer (DDS) RFcomb generator.84. The integrated circuit according to any one of clauses 81-83,wherein the light beam generator component is configured to generate afrequency-shifted local oscillator beam.85. The integrated circuit according to any one of clauses 80-84,wherein the interferometric phase adjustment comprises a phase shiftresulting from vibrations between components of the light source.86. The integrated circuit according any one of clauses 76-85, whereinthe integrated circuit is programmed to calculate the second phaseadjustment based on a fluorescence lifetime of a fluorophore in thesample.87. The integrated circuit according to any one of clauses 69-86,wherein the integrated circuit is a field programmable gate array(FPGA).88. The integrated circuit according to any one of clauses 69-86,wherein the integrated circuit device is an application specificintegrated circuit (ASIC).89. The integrated circuit according to clause 69-86, wherein theintegrated circuit device is a complex programmable logic device (CPLD).90. The integrated circuit according to any one of clauses 69-89,wherein the integrated circuit is programmed to generate an image of theparticle from the phase-corrected spatial data.91. The integrated circuit according to clause 90, wherein theintegrated circuit is programmed to generate an image mask of theparticle.92. The integrated circuit according to any one of clauses 69-91,wherein the integrated circuit is programmed to generate a sortingdecision based on the phase-corrected spatial data.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. Moreover, nothing disclosedherein is intended to be dedicated to the public regardless of whethersuch disclosure is explicitly recited in the claims.

The scope of the present invention, therefore, is not intended to belimited to the exemplary embodiments shown and described herein. Rather,the scope and spirit of present invention is embodied by the appendedclaims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) isexpressly defined as being invoked for a limitation in the claim onlywhen the exact phrase “means for” or the exact phrase “step for” isrecited at the beginning of such limitation in the claim; if such exactphrase is not used in a limitation in the claim, then 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is not invoked.

1. A method comprising: generating frequency-encoded fluorescence datafrom a particle of a sample in a flow stream; and calculatingphase-corrected spatial data of the particle by performing a transformof the frequency-encoded fluorescence data with a phase correctioncomponent.
 2. The method according to claim 1, wherein the spatial datais calculated by performing a Fourier transform of the frequency-encodedfluorescence data with the phase correction component.
 3. The methodaccording to claim 2, wherein the spatial data is calculated byperforming a discrete Fourier transform of the frequency-encodedfluorescence data with the phase correction component.
 4. The methodaccording to claim 2, wherein the spatial data is calculated byperforming a short time Fourier transform (STFT) of thefrequency-encoded fluorescence data with the phase correction component.5. The method according to claim 1, wherein the spatial data iscalculated with a digital lock-in amplifier to heterodyne andde-multiplex the frequency-encoded fluorescence data.
 6. The methodaccording to claim 1, wherein the phase correction component comprisesmodified transform coefficients that are used to transform thefrequency-encoded fluorescence data into the phase-corrected spatialdata.
 7. The method according to claim 1, wherein generating thefrequency-encoded fluorescence data from the particle comprisesdetecting light from the particle in the sample with a light detectionsystem.
 8. The method according to claim 7, wherein the light detectedfrom the particle comprises light absorption, light scatter, emittedlight or a combination thereof. 9.-10. (canceled)
 11. The methodaccording to claim 1, wherein the phase correction component comprises afirst phase adjustment and a second phase adjustment.
 12. The methodaccording to claim 11, wherein the first phase adjustment comprises anoutput signal from the light detection system.
 13. The method accordingto claim 12, wherein the first phase adjustment comprises an outputsignal from a brightfield photodetector.
 14. The method according toclaim 13, further comprising calculating the first phase adjustment by:multiplying an output signal from the brightfield photodetector with apredetermined constant signal to produce a phase adjustment value; andcalculating the arctangent of the phase adjustment value to generate thefirst phase adjustment.
 15. The method according to claim 14, whereinthe phase adjustment value is a sum of all bins in a discrete Fouriertransform of the frequency-encoded fluorescence data.
 16. The methodaccording to claim 11, wherein the first phase adjustment is aninterferometric phase adjustment. 17.-24. (canceled)
 25. The methodaccording to claim 11, further comprising calculating the second phaseadjustment based on a fluorescence lifetime of a fluorophore in thesample.
 26. The method according to claim 1, wherein the phase-correctedspatial data of the particle is calculated from the frequency-encodedfluorescence data by an integrated circuit device. 27.-35. (canceled)36. The method according to claim 1, further comprising generating animage of the particle from the phase-corrected spatial data. 37.(canceled)
 38. The method according to claim 1, further comprisingsorting the particle.
 39. A system comprising: a light source configuredto irradiate a sample comprising particles in a flow stream; a lightdetection system; and a processor comprising memory operably coupled tothe processor wherein the memory comprises instructions stored thereon,which when executed by the processor, cause the processor to: generatefrequency-encoded fluorescence data from a particle in the flow stream;calculate phased-corrected spatial data of the particle by performing atransform of the frequency-encoded fluorescence data with a phasecorrection component. 40.-69. (canceled)
 70. An integrated circuitprogrammed to: generate frequency-encoded fluorescence data from aparticle in the flow stream; calculate phase-corrected spatial data ofthe particle by performing a transform of the frequency-encodedfluorescence data with a phase correction component. 71.-92. (canceled)